https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Travis+EyfordProteopedia - User contributions [en]2026-09-23T20:16:32ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=Adenylyl_cyclase&diff=1077056Adenylyl cyclase2010-04-16T00:37:23Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_1y11| PDB=1y11 | SCENE=Sandbox_159/Main_1y11/1 }}<br />
Adenylyl cyclase, also known as adenylate cyclase, is an enzyme which catalyzes the cyclization of [http://en.wikipedia.org/wiki/Adenosine_triphosphate adenosine triphosphate] (ATP) into [http://en.wikipedia.org/wiki/Cyclic_adenosine_monophosphate cyclic adenosine monophosphate] (cAMP) which requires the cleavage of [http://en.wikipedia.org/wiki/Pyrophosphate pyrophosphate] (PPi)<ref name="Taussig">PMID:7814360</ref>. This page emphasizes on the microbial adenylyl cyclase Rv1264, but mammalian adenylyl cyclases are also covered in less detail.<br />
<br />
== Introduction ==<br />
There are ten isozymes of adenylyl cyclases in mammals, adenylyl cyclase type I-X, (ADCY I-X), and many more in other organisms<ref name="Taussig"/>. All mammalian, and most other adenylyl cyclases belong to class III; most are integral membrane proteins, and all produce cAMP, the ability of which can be activated or inactivated in response to certain conditions or ligands<ref name="Taussig"/>. All mammalian membrane bound adenylyl cyclases are activated by alpha subunits of [http://en.wikipedia.org/wiki/G-protein/ G-proteins], but respond differently to ligands such as magnesium ions, calcium ions, and beta gamma subunits of G proteins<ref name="Taussig"/>. One of the mammalian isozymes, and some prokaryotic forms of adenylyl cyclase respond to environmental conditions, primarily pH<ref>PMID:14512417</ref><ref name="Linder">PMID:11839758</ref>.<br />
<br />
== Reaction ==<br />
=== Reactant ===<br />
The reactant in the reaction catalyzed by adenylyl cyclase is ATP; ATP is the most abundant nucleotide triphosphate in most cells with typical concentrations ranging from 1 to 10mM<ref>PMID:1212224</ref>. This high intracellular concentration allows for cAMP concentrations to rise quickly in response to a specific signal, which is important in many signal transduction and metabolic pathways<ref>PMID:182581</ref>.<br />
<br />
=== Reaction ===<br />
[[Image:CAMP synthesis.png|frame|center]]<br />
The reaction occurs in a single, concerted step, where the oxygen on ATP's 3' hydroxyl group nucleophillically attacks the alpha-phosphate forming a phosphodiester bond and cleaving a pyrophosphate group<ref name="Hurley">PMID:10075642</ref>. In most active sites there is an acidic residue near the 3'OH which functions in its deprotonation, and basic residues by the β-phosphorous to lower the energy of the group for cleavage<ref name="Zhang">PMID:9069282</ref><ref>PMID:14747729</ref>.<br />
<br />
=== Products ===<br />
The main product of this reaction is cAMP, with a side product of PP<sub>i</sub><ref name="Taussig"/>.<br />
==== Cyclic Adenosine Monophosphate ====<br />
In mammals, cAMP acts as a secondary messenger, one of its functions is to control the activity of [http://en.wikipedia.org/wiki/Protein_kinase_A protein kinase A] (PKA)<ref name="Siddappa">PMID:18490653</ref>. In turn, PKA has quite diverse roles in cells, while most of them are associated with metabolism, PKA also plays important roles in transcription, the cell cycle, and [http://en.wikipedia.org/wiki/Apoptosis apoptosis]<ref name="Siddappa"/>. The ultimate fate of cAMP is its transformation into AMP by the cleavage of the phosphodiester bond by 3', 5'-cyclic adenosine monophosphate phosphodiesterase<ref name="Siddappa"/>.<br />
<br />
==== Pyrophosphate ====<br />
Cleavage of the by-product of this reaction, PP<sub>i</sub>, by pyrophosphatase yields two molecules of [http://en.wikipedia.org/wiki/Phosphate inorganic phosphate] (P<sub>i</sub>)<ref>PMID:4964763</ref>. ATP synthase can reincorporate this inorganic phosphate into adenine diphosphate (ADP) to make ATP using energy in the proton motive force<ref>PMID:9242922</ref>.<br />
<br />
== Mammalian Adenylyl Cyclase ==<br />
There are ten isozymes of adenylyl cyclases in mammals, adenylyl cyclase type I-X, (ADCY I-X); In mammals adenylyl cyclase plays an important role in signal transduction pathways in which cAMP is a secondary messenger<ref name="Feinstein">PMID:1719547</ref>.<br />
<br />
ADCY I-IX all share a general structure; They are composed of two trans-membrane regions (M1, M2) which are composed of six membrane-spanning helices and function to keep the enzyme anchored in the membrane, and two cytoplasmic regions (C<sub>1</sub>, C<sub>2</sub>) which can be further sub divided (C<sub>1a</sub>, C<sub>1b</sub>, C<sub>2a</sub>, C<sub>2b</sub>) and are responsible for all catalytic activity, and regulation by G-proteins and [http://en.wikipedia.org/wiki/Forskolin forskolin]<ref name="Feinstein"/>. In solution, the C<sub>1a</sub> and C<sub>2a</sub> domains can form heterodimers with each other, either in the same or different enzymes, or they can form homodimers with their identical units on different enzymes<ref name="Zhang"/>. The C<sub>1b</sub> domain is very large (≈15 kDa) with many regulatory sites, and has a variable structure across isozymes; while the C<sub>2b</sub> domain is nearly non-existent in many isozymes, and has yet to be associated with a particular function<ref name="Zhang"/>. <br />
=== Type II ===<br />
==== Structure ====<br />
<applet load='1ab8' size='300' frame='true' align='right' caption='Type II adenylyl cyclase homodimer with two bound forskolin molecules' scene='Sandbox_159/Full_structure_1ab8/1'/><br />
A monomer of C<sub>2</sub> domain of type II adenylyl cyclase has an internal, hydrophobic, anti-parallel <scene name='Sandbox_159/Beta_sheets_1ab8/1'>β-sheet</scene> surrounded by several, amphipathic <scene name='Sandbox_159/Helix_1ab8/1'>α-helices</scene>, except for an area which needed to form a homodimer with another C<sub>2</sub> domain<ref name="Zhang"/>. Two monomers of C<sub>2</sub> domains of type II adenylyl cyclase bind together in solution to form a <scene name='Sandbox_159/Full_structure_1ab8/1'>Homodimer</scene>, which is necessary for catalytic conversion of ATP to cAMP and PPi<ref name="Hurley"/>. When they are bound they create a deep crevasse spanning the center of their binding site; this crevasse is suited to bind two <scene name='Sandbox_159/Forskolin_1ab8/4'>forskolin</scene> molecules at its ends<ref name="Tesmer">PMID:9417641</ref>. Strong hydrogen bonds are made between oxygen atoms of forskolin and the surrounding peptide backbone, and the rest of the interactions are highly hydrophobic, as the forskolin binding site contains ten aliphatic and aromatic residues<ref name="Tesmer"/>. This binding of forskolin creates a hydrophobic linkage between the monomers, each of which has two different hydrophobic surfaces binding to forskolin; and it is this interaction which makes the homodimer stable<ref name="Zhang"/><ref name="Tesmer"/>. The forskolin also interacts with and properly positions Asn 1025, which is essential for catalytic activity, and it may even interact directly with the ATP<ref name="Tesmer"/>. This homodimer-forskolin complex can be further activated in response to a signal via binding to a G-protein’s βγ-subunit <ref name="Hurley"/><ref name="Tesmer"/>. This βγ-subunit binds to <scene name='Sandbox_159/G-protein_binding_site/1'>residues 956 to 982</scene> which make up part of an α-helix on the outermost layer of the complex<ref name="Hurley"/><ref>PMID:2899356</ref>. <br />
<br />
The <scene name='Sandbox_159/Activesite_1ab8/3'>active site</scene> of this homodimer is located within the crevasse, and is characterized by two highly conserved sets of polar residues (Arg 997 (Green), Asn 1025 (Red), Ser1028(Pink), Arg 1029(Orange), Asp 1031(Yellow), and Ser 1032(Purple))<ref name="Hurley"/><ref name="Zhang"/>. One of these sets is located on each monomeric subunit, on the homodimer they arrange themselves in an anti-parallel fashion, where they point towards each other<ref name="Zhang"/>.<br />
<br />
==== Over-expression Disorders ====<br />
In the brain of mammals, the execution of memory based functions is carried out by the [http://en.wikipedia.org/wiki/Prefrontal_cortex prefrontal cortex] (PFC)<ref name="Wang">PMID:17448997</ref>. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels on neurons close to allow electrochemical signals to flow down the axon and into a synapse; when HCN channels are open, the electropotential signal cannot be transmitted through the cell<ref name="Wang"/>. Exposure of these channels to cAMP causes them open, stopping the transmission of signals, and thus impairs higher cognitive thoughts<ref name="Wang"/>. In patients with schizophrenia, a cAMP regulatory molecule, [http://en.wikipedia.org/wiki/DISC1 Disrupted-in-Schizophrenia 1] (DISC1) is mutated and cannot regulate cAMP levels<ref name="Wang"/>; Thus, elevated cAMP levels may cause schizophrenia. The closing of HCN channels are thought to play a part in other disorders, such as ADHD and bipolar disorder<ref name="Wang"/>. Thus, it is reasonable that regulation of cAMP production by targeting type II adenylyl cyclase, since it is found in the brain, may act as a treatment for these disorders.<br />
<br />
== Rv1264 Adenylyl Cyclase ==<br />
Although adenylyl cyclase is found throughout organisms at a universal level, distantly related organisms have different modifications of the enzyme, each is specialized for a particular task in a particular environment<ref name="Tews">PMID:15890882</ref>. As stated earlier humans have 10 known isozymes of adenylyl cyclase; whereas ''Escherichia coli'' has only one isozyme, and ''Mycobacterium tuberculosis'' has 15<ref name="Tews"/>. One particularly interesting adenylyl cyclase possessed by ''M. tuberculosis'', Rv1264, has a N-terminal which, in a sense, acts as a pH sensor, as it regulates the activity of the enzyme based on the pH of the surrounding solution<ref name="Linder"/><ref name="Tews"/>. This adenylyl cyclase, like most others, belongs to class III, adenylyl cyclases in this class have multiple domains, at least one for catalysis, and another for regulation<ref name="Tews"/>.<br />
=== Structure ===<br />
<applet load='1y11' size='380' frame='true' align='right' caption='Rv1264 adenylyl cyclase monomer in its active state.' scene='Sandbox_159/Main_1y11/2'/><br />
The <scene name='Sandbox_159/Main_1y11/2'>Rv1264</scene> adenylyl cyclase is a 363 residue long protein composed of a catalytic <scene name='Sandbox_159/Cdomain_1y11/1'>C-terminal domain</scene> and a regulatory <scene name='Sandbox_159/Ndomain_1y11/2'>N-terminal domain</scene> which contains a flexible <scene name='Sandbox_159/Connector_1y11/2'>linker region</scene> which connects the two<ref name="Tews"/>. The active structure is a homodimer resembling the mammalian type II homodimer, where an α-helix of one monomer (Chain A) is placed through the central coiled coil of another (Chain B)<ref name="Zhang"/><ref name="Tews"/>. This dimerization places the regulatory domain of chain A in close proximity to the catalytic domain of chain B, and vice versa<ref name="Tews"/>. Two switch elements are present in the protein, one in the C-terminal domain (α1-switch) and another in the linker region (αN10-switch), these allow for large conformational changes to take place in the enzyme in response to relatively small environmental changes<ref name="Linder"/><ref name="Tews"/>. <br />
==== C-Terminal Catalytic Domain ====<br />
The catalytic activity in Rv1264's <scene name='Sandbox_159/Active_1y11/1'>active site</scene> is performed by the residues: Asp 222 (Red), Lys 261 (Blue), Asp 265 (Orange), Arg 298 (Pink), Asp 312 (Yellow), Asn 319 (Purple), Arg 323 (Green)<ref name="Tews"/>. All of these residues create a highly polar environment which is complementary in charge and polarity to the intermediate of the reaction<ref name="Linder"/><ref name="Tews"/>. Residues which guide the phosphates of ATP, arginine 298 and 323, bind a <scene name='Sandbox_159/Sulphate_1y11/1'>sulphate ion</scene> in the active site<ref name="Tesmer2">PMID:10427002</ref>. This sulphate ion is located in the position which will be occupied by the ATP's β-phosphate during catalysis<ref name="Tesmer2"/>. In the process of catalysis the β-phosphate is cleaved from the α-phosphate; this reaction may be made more favourable by lowering the energy though complementary charge associations between the β-phosphate and arginines 298 and 323<ref name="Tews"/><ref name="Tesmer2"/>. Another residue, <scene name='Sandbox_159/Argglycerol_1y11/1'>arginine 296</scene>, binds a <scene name='Sandbox_159/Glycerol_1y11/1'>glycerol</scene> molecule through electrostatic interactions<ref name="Tews"/>. The specific function of this association is unknown, but because of its close proximity to the active site it may play a role in catalysis<ref name="Tews"/>. <br />
<br />
Amino acid sequencing has shown that the sequence between the Rv1264 adenylyl cyclases catalytic domain and mammalian adenylyl cyclases catalytic domain are not well conserved, with only about a 25% correspondence to the mammalian type II adenylyl cyclase discussed above<ref name="Linder"/><ref name="Tews"/>. The two different isozymes; however, still resemble each other in that superimposition of one over the other has a substantial overlap, with a [http://en.wikipedia.org/wiki/Root_mean_square_deviation root mean square deviation] (rmsd) of less than 1.76Å between 79% of all <scene name='Sandbox_159/Alphac_1y11/1'>α-carbons</scene><ref name="Linder"/><ref name="Tews"/>. A notable difference in Rv1264 and type II adenylyl cyclase catalytic domains is their relative sizes; Rv1264 has no dimerization arm and several of its loops have been shortened<ref name="Linder"/><ref name="Tews"/>. This results in Rv1264 adenylyl cyclase catalytic domain associating as a dimer with a smaller [http://en.wikipedia.org/wiki/Interface_(chemistry) interface] area than mammal's type II, with interface areas of 1900Å<sup>2</sup> and 3800Å<sup>2</sup>, respectively<ref name="Tews"/>. The active sites the Rv1264 and mammalian type II adenylyl cyclases are even more conserved; the <scene name='Sandbox_159/Activealpha_1y11/2'>α-carbons</scene> in the active site have an rmsd of only 0.69Å, and <scene name='Sandbox_159/Activeatoms_1y11/2'>all atoms</scene> in the active site have a rmsd of 1.17Å<ref name="Linder"/><ref name="Tews"/>.<br />
[[Image:Dimer2.png|frame|left|Rv1264 dimer in its active state.]]<br />
All of the above structural information for the C-terminal catalytic domain is only true when the enzyme is in its active state. The inactive state of the enzyme possesses a disassembled active site, hence no catalytic activity. Relative to the fixed N-terminal domains, each of the C-terminal monomeric domains can transpose up to 6Å and rotate by 55<sup>o</sup><ref name="Tews"/>. This massive change in the C-terminal domains tertiary structure disassembles the active site, moving catalytic residues up to 25Å away from their catalytically active position<ref name="Tews"/>. Not only is the active site disrupted in the C-terminal domain, but the interface area between the two monomers decreases in size from 1900Å<sup>2</sup> to 930Å<sup>2</sup><ref name="Tews"/>.<br />
===== α1-switch =====<br />
The <scene name='Sandbox_159/Alphaoneswitch_1y11/1'>α1-switch</scene> is located within the C-terminal catalytic domain and exists as a compact α-helix when the enzyme is in its active state,<ref name="Tews"/>. Upon inactivation the α-helical conformation of the α1-switch becomes unstable, and it transforms into a random coil<ref name="Tews"/>. Since this switch is in a close proximity to the active site, a major change in structure greatly disrupts the active site, and yields the enzyme inactive<ref name="Tews"/>.<br />
<br />
This structure is conserved in mammalian adenylyl cyclases, where it acts as a contributor for the binding site of the βγ-phosphates of the ATP substrate<ref name="Tesmer3">PMID:11087399</ref>. It also functions in regulation of mammalian adenylyl cyclases, along with another α-helix, it forms the binding site for the G<sub>s</sub>α and G<sub>i</sub>α G protein subunits which regulates the adenylyl cyclases activity<ref name="Zhang"/>.<br />
<br />
==== N-Terminal Regulatory Domain ====<br />
<applet load='1y11' size='380' frame='true' align='right' caption='Rv1264 adenylyl cyclase in its active state.' scene='Sandbox_159/Main_1y11/2'/><br />
<br />
The N-terminal domain functions in regulation; it has a unique mechanism that determines whether the enzyme will be active or not based on the pH of the surrounding solution<ref name="Linder"/>. Each monomer in the catalytically active dimer has ten <scene name='Sandbox_159/Ntermhelix_1y11/1'>α-helices</scene>, when they are dimerized they form a disc like structure<ref name="Tews"/>. A single molecule of <scene name='Sandbox_159/Peg_1y11/1'>pentaethylene glycol</scene> binds in a hydrophobic pocket<ref name="Findeisen">PMID:17482646</ref>. The function of this polyethylene glycol appears to be structural; however, its specific placement and movement between active and inactive forms of the enzyme suggests that it may also function in detecting hydrophobicty changes in the environment<ref name="Findeisen"/>.<br />
===== αN10-switch =====<br />
The <scene name='Sandbox_159/Linkerswitch_1y11/1'>αN10-switch</scene> is located within the linker region and its conformational change has a more drastic effect on the overall enzyme than the α1-switch does<ref name="Tews"/><ref name="Findeisen"/>. When the enzyme is active, the αN10-switch consists of a random coil with a short α-helix; this conformation allows for only a weak interaction between the N-terminal regulatory domain and the C-terminal catalytic domain, allowing the enzyme to exhibit catalytic activity<ref name="Tews"/>. This helix can extend by up to 24Å, which separates the monomeric C-terminal catalytic domains of the dimer<ref name="Tews"/>. This separation of domains lowers their interface area and transposes residues; as discussed above, these changes result in an inactivation of the enzyme, and thus, a major characteristic of the inactive state of the enzyme is an extended αN10-switch<ref name="Tews"/><ref name="Findeisen"/>. As the αN10-switch extends, the <scene name='Sandbox_159/Alpha4_1y11/1'>α4-helix</scene> moves outwards, and the pentaethylene glycol moves into a newly formed cavity by the αN10 helix<ref name="Findeisen"/>. This further supports the idea that the pentaethylene glycol ligand functions not only for structure, but also for regulation<ref name="Findeisen"/>.<br />
<br />
=== Regulation by pH ===<br />
At the beginning of the linker region there is a residue, <scene name='Sandbox_159/His192_1y11/1'>His 192</scene>, which has a considerable influence on regulation by pH<ref name="Tews"/><ref name="Findeisen"/>. At a basic pH the residue has no charge and minimal interactions with the two catalytically important residues <scene name='Sandbox_159/Lysargactive_1y11/2'>Lys 261 (green) and Asp 312 (red)</scene>, which lie 14 Å and 21 Å away from their catalytically active positions<ref name="Tews"/><ref name="Findeisen"/>. The enzyme is inactive at this state, not only because of the Lys 261 and Asp 312 residues, but also because of other major structural components of the catalytic site being disassembled. At an acidic pH Rv1264 becomes active (Optimal pH~5.8)and has up to a 40 fold increase in activity<ref name="Tews"/>. At this acidic pH, His 192 becomes protonated and positively charged; which creates major structural changes throughout the protein, including compaction of both the αN10-switch and α1-switch, contraction of the α4-helix which in turn causes a translocation of the paraethylene glycol ligand<ref name="Linder"/><ref name="Tews"/><ref name="Findeisen"/>. The positively charged His 192 electrostatically repels the Lys 261 and Asp 312 residues which along with other structural changes transposes them 14 Å and 21 Å, respectively, into their catalytically active positions<ref name="Tews"/>.<br />
<br />
<scene name='Sandbox_159/Arg309_1y11/1'>Arginine 309</scene>, a residue which organizes many residues which are important in the C-terminal - N-terminal interaction through hydrogen bonds, is also important for regulation<ref name="Tews"/>. When this residue is mutated, regulation based on pH is lost and the enzyme is constantly active<ref name="Tews"/>.<br />
<br />
Many other residues also contribute to regulation by pH; it is the electrostatic interactions and hydrogen bonds in the C-terminal - N-terminal domain interface which allows Rv1264 adenylyl cyclase to be sensitive to pH<ref name="Tews"/>.<br />
<br />
=== Role ===<br />
''M. tuberculosis'' is a pathogenic bacterium, and thus it faces an array of a host's immune responses to attempt in an attempt to rid of it<ref>PMID:11239406</ref>. One of the hosts defense mechanisms ''M. tuberculosis'' faces is acidification encountered in phagolysosomes. The ability to be able to detect this acidic environment, and have an appropriate response to it may greatly assist ''M. tuberculosis'' infect a host<ref>PMID:15275372</ref><ref>PMID:17342138</ref>. As cAMP levels are increased, acidification of other structures is delayed and elevated cAMP levels activate [http://en.wikipedia.org/wiki/CAMP_receptor_protein cAMP receptor proteins] which in turn regulate transcription<ref>PMID:165421</ref><ref>PMID:15882420</ref>.<br />
<br />
== References ==<br />
<references/></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Adenylyl_cyclase&diff=1077055Adenylyl cyclase2010-04-16T00:35:38Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_1y11| PDB=1y11 | SCENE=Sandbox_159/Main_1y11/1 }}<br />
Adenylyl cyclase, also known as adenylate cyclase, is an enzyme which catalyzes the cyclization of [http://en.wikipedia.org/wiki/Adenosine_triphosphate adenosine triphosphate] (ATP) into [http://en.wikipedia.org/wiki/Cyclic_adenosine_monophosphate cyclic adenosine monophosphate] (cAMP) which requires the cleavage of [http://en.wikipedia.org/wiki/Pyrophosphate pyrophosphate] (PPi)<ref name="Taussig">PMID:7814360</ref>.<br />
<br />
== Introduction ==<br />
There are ten isozymes of adenylyl cyclases in mammals, adenylyl cyclase type I-X, (ADCY I-X), and many more in other organisms<ref name="Taussig"/>. All mammalian, and most other adenylyl cyclases belong to class III; most are integral membrane proteins, and all produce cAMP, the ability of which can be activated or inactivated in response to certain conditions or ligands<ref name="Taussig"/>. All mammalian membrane bound adenylyl cyclases are activated by alpha subunits of [http://en.wikipedia.org/wiki/G-protein/ G-proteins], but respond differently to ligands such as magnesium ions, calcium ions, and beta gamma subunits of G proteins<ref name="Taussig"/>. One of the mammalian isozymes, and some prokaryotic forms of adenylyl cyclase respond to environmental conditions, primarily pH<ref>PMID:14512417</ref><ref name="Linder">PMID:11839758</ref>.<br />
<br />
== Reaction ==<br />
=== Reactant ===<br />
The reactant in the reaction catalyzed by adenylyl cyclase is ATP; ATP is the most abundant nucleotide triphosphate in most cells with typical concentrations ranging from 1 to 10mM<ref>PMID:1212224</ref>. This high intracellular concentration allows for cAMP concentrations to rise quickly in response to a specific signal, which is important in many signal transduction and metabolic pathways<ref>PMID:182581</ref>.<br />
<br />
=== Reaction ===<br />
[[Image:CAMP synthesis.png|frame|center]]<br />
The reaction occurs in a single, concerted step, where the oxygen on ATP's 3' hydroxyl group nucleophillically attacks the alpha-phosphate forming a phosphodiester bond and cleaving a pyrophosphate group<ref name="Hurley">PMID:10075642</ref>. In most active sites there is an acidic residue near the 3'OH which functions in its deprotonation, and basic residues by the β-phosphorous to lower the energy of the group for cleavage<ref name="Zhang">PMID:9069282</ref><ref>PMID:14747729</ref>.<br />
<br />
=== Products ===<br />
The main product of this reaction is cAMP, with a side product of PP<sub>i</sub><ref name="Taussig"/>.<br />
==== Cyclic Adenosine Monophosphate ====<br />
In mammals, cAMP acts as a secondary messenger, one of its functions is to control the activity of [http://en.wikipedia.org/wiki/Protein_kinase_A protein kinase A] (PKA)<ref name="Siddappa">PMID:18490653</ref>. In turn, PKA has quite diverse roles in cells, while most of them are associated with metabolism, PKA also plays important roles in transcription, the cell cycle, and [http://en.wikipedia.org/wiki/Apoptosis apoptosis]<ref name="Siddappa"/>. The ultimate fate of cAMP is its transformation into AMP by the cleavage of the phosphodiester bond by 3', 5'-cyclic adenosine monophosphate phosphodiesterase<ref name="Siddappa"/>.<br />
<br />
==== Pyrophosphate ====<br />
Cleavage of the by-product of this reaction, PP<sub>i</sub>, by pyrophosphatase yields two molecules of [http://en.wikipedia.org/wiki/Phosphate inorganic phosphate] (P<sub>i</sub>)<ref>PMID:4964763</ref>. ATP synthase can reincorporate this inorganic phosphate into adenine diphosphate (ADP) to make ATP using energy in the proton motive force<ref>PMID:9242922</ref>.<br />
<br />
== Mammalian Adenylyl Cyclase ==<br />
There are ten isozymes of adenylyl cyclases in mammals, adenylyl cyclase type I-X, (ADCY I-X); In mammals adenylyl cyclase plays an important role in signal transduction pathways in which cAMP is a secondary messenger<ref name="Feinstein">PMID:1719547</ref>.<br />
<br />
ADCY I-IX all share a general structure; They are composed of two trans-membrane regions (M1, M2) which are composed of six membrane-spanning helices and function to keep the enzyme anchored in the membrane, and two cytoplasmic regions (C<sub>1</sub>, C<sub>2</sub>) which can be further sub divided (C<sub>1a</sub>, C<sub>1b</sub>, C<sub>2a</sub>, C<sub>2b</sub>) and are responsible for all catalytic activity, and regulation by G-proteins and [http://en.wikipedia.org/wiki/Forskolin forskolin]<ref name="Feinstein"/>. In solution, the C<sub>1a</sub> and C<sub>2a</sub> domains can form heterodimers with each other, either in the same or different enzymes, or they can form homodimers with their identical units on different enzymes<ref name="Zhang"/>. The C<sub>1b</sub> domain is very large (≈15 kDa) with many regulatory sites, and has a variable structure across isozymes; while the C<sub>2b</sub> domain is nearly non-existent in many isozymes, and has yet to be associated with a particular function<ref name="Zhang"/>. <br />
=== Type II ===<br />
==== Structure ====<br />
<applet load='1ab8' size='300' frame='true' align='right' caption='Type II adenylyl cyclase homodimer with two bound forskolin molecules' scene='Sandbox_159/Full_structure_1ab8/1'/><br />
A monomer of C<sub>2</sub> domain of type II adenylyl cyclase has an internal, hydrophobic, anti-parallel <scene name='Sandbox_159/Beta_sheets_1ab8/1'>β-sheet</scene> surrounded by several, amphipathic <scene name='Sandbox_159/Helix_1ab8/1'>α-helices</scene>, except for an area which needed to form a homodimer with another C<sub>2</sub> domain<ref name="Zhang"/>. Two monomers of C<sub>2</sub> domains of type II adenylyl cyclase bind together in solution to form a <scene name='Sandbox_159/Full_structure_1ab8/1'>Homodimer</scene>, which is necessary for catalytic conversion of ATP to cAMP and PPi<ref name="Hurley"/>. When they are bound they create a deep crevasse spanning the center of their binding site; this crevasse is suited to bind two <scene name='Sandbox_159/Forskolin_1ab8/4'>forskolin</scene> molecules at its ends<ref name="Tesmer">PMID:9417641</ref>. Strong hydrogen bonds are made between oxygen atoms of forskolin and the surrounding peptide backbone, and the rest of the interactions are highly hydrophobic, as the forskolin binding site contains ten aliphatic and aromatic residues<ref name="Tesmer"/>. This binding of forskolin creates a hydrophobic linkage between the monomers, each of which has two different hydrophobic surfaces binding to forskolin; and it is this interaction which makes the homodimer stable<ref name="Zhang"/><ref name="Tesmer"/>. The forskolin also interacts with and properly positions Asn 1025, which is essential for catalytic activity, and it may even interact directly with the ATP<ref name="Tesmer"/>. This homodimer-forskolin complex can be further activated in response to a signal via binding to a G-protein’s βγ-subunit <ref name="Hurley"/><ref name="Tesmer"/>. This βγ-subunit binds to <scene name='Sandbox_159/G-protein_binding_site/1'>residues 956 to 982</scene> which make up part of an α-helix on the outermost layer of the complex<ref name="Hurley"/><ref>PMID:2899356</ref>. <br />
<br />
The <scene name='Sandbox_159/Activesite_1ab8/3'>active site</scene> of this homodimer is located within the crevasse, and is characterized by two highly conserved sets of polar residues (Arg 997 (Green), Asn 1025 (Red), Ser1028(Pink), Arg 1029(Orange), Asp 1031(Yellow), and Ser 1032(Purple))<ref name="Hurley"/><ref name="Zhang"/>. One of these sets is located on each monomeric subunit, on the homodimer they arrange themselves in an anti-parallel fashion, where they point towards each other<ref name="Zhang"/>.<br />
<br />
==== Over-expression Disorders ====<br />
In the brain of mammals, the execution of memory based functions is carried out by the [http://en.wikipedia.org/wiki/Prefrontal_cortex prefrontal cortex] (PFC)<ref name="Wang">PMID:17448997</ref>. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels on neurons close to allow electrochemical signals to flow down the axon and into a synapse; when HCN channels are open, the electropotential signal cannot be transmitted through the cell<ref name="Wang"/>. Exposure of these channels to cAMP causes them open, stopping the transmission of signals, and thus impairs higher cognitive thoughts<ref name="Wang"/>. In patients with schizophrenia, a cAMP regulatory molecule, [http://en.wikipedia.org/wiki/DISC1 Disrupted-in-Schizophrenia 1] (DISC1) is mutated and cannot regulate cAMP levels<ref name="Wang"/>; Thus, elevated cAMP levels may cause schizophrenia. The closing of HCN channels are thought to play a part in other disorders, such as ADHD and bipolar disorder<ref name="Wang"/>. Thus, it is reasonable that regulation of cAMP production by targeting type II adenylyl cyclase, since it is found in the brain, may act as a treatment for these disorders.<br />
<br />
== Rv1264 Adenylyl Cyclase ==<br />
Although adenylyl cyclase is found throughout organisms at a universal level, distantly related organisms have different modifications of the enzyme, each is specialized for a particular task in a particular environment<ref name="Tews">PMID:15890882</ref>. As stated earlier humans have 10 known isozymes of adenylyl cyclase; whereas ''Escherichia coli'' has only one isozyme, and ''Mycobacterium tuberculosis'' has 15<ref name="Tews"/>. One particularly interesting adenylyl cyclase possessed by ''M. tuberculosis'', Rv1264, has a N-terminal which, in a sense, acts as a pH sensor, as it regulates the activity of the enzyme based on the pH of the surrounding solution<ref name="Linder"/><ref name="Tews"/>. This adenylyl cyclase, like most others, belongs to class III, adenylyl cyclases in this class have multiple domains, at least one for catalysis, and another for regulation<ref name="Tews"/>.<br />
=== Structure ===<br />
<applet load='1y11' size='380' frame='true' align='right' caption='Rv1264 adenylyl cyclase monomer in its active state.' scene='Sandbox_159/Main_1y11/2'/><br />
The <scene name='Sandbox_159/Main_1y11/2'>Rv1264</scene> adenylyl cyclase is a 363 residue long protein composed of a catalytic <scene name='Sandbox_159/Cdomain_1y11/1'>C-terminal domain</scene> and a regulatory <scene name='Sandbox_159/Ndomain_1y11/2'>N-terminal domain</scene> which contains a flexible <scene name='Sandbox_159/Connector_1y11/2'>linker region</scene> which connects the two<ref name="Tews"/>. The active structure is a homodimer resembling the mammalian type II homodimer, where an α-helix of one monomer (Chain A) is placed through the central coiled coil of another (Chain B)<ref name="Zhang"/><ref name="Tews"/>. This dimerization places the regulatory domain of chain A in close proximity to the catalytic domain of chain B, and vice versa<ref name="Tews"/>. Two switch elements are present in the protein, one in the C-terminal domain (α1-switch) and another in the linker region (αN10-switch), these allow for large conformational changes to take place in the enzyme in response to relatively small environmental changes<ref name="Linder"/><ref name="Tews"/>. <br />
==== C-Terminal Catalytic Domain ====<br />
The catalytic activity in Rv1264's <scene name='Sandbox_159/Active_1y11/1'>active site</scene> is performed by the residues: Asp 222 (Red), Lys 261 (Blue), Asp 265 (Orange), Arg 298 (Pink), Asp 312 (Yellow), Asn 319 (Purple), Arg 323 (Green)<ref name="Tews"/>. All of these residues create a highly polar environment which is complementary in charge and polarity to the intermediate of the reaction<ref name="Linder"/><ref name="Tews"/>. Residues which guide the phosphates of ATP, arginine 298 and 323, bind a <scene name='Sandbox_159/Sulphate_1y11/1'>sulphate ion</scene> in the active site<ref name="Tesmer2">PMID:10427002</ref>. This sulphate ion is located in the position which will be occupied by the ATP's β-phosphate during catalysis<ref name="Tesmer2"/>. In the process of catalysis the β-phosphate is cleaved from the α-phosphate; this reaction may be made more favourable by lowering the energy though complementary charge associations between the β-phosphate and arginines 298 and 323<ref name="Tews"/><ref name="Tesmer2"/>. Another residue, <scene name='Sandbox_159/Argglycerol_1y11/1'>arginine 296</scene>, binds a <scene name='Sandbox_159/Glycerol_1y11/1'>glycerol</scene> molecule through electrostatic interactions<ref name="Tews"/>. The specific function of this association is unknown, but because of its close proximity to the active site it may play a role in catalysis<ref name="Tews"/>. <br />
<br />
Amino acid sequencing has shown that the sequence between the Rv1264 adenylyl cyclases catalytic domain and mammalian adenylyl cyclases catalytic domain are not well conserved, with only about a 25% correspondence to the mammalian type II adenylyl cyclase discussed above<ref name="Linder"/><ref name="Tews"/>. The two different isozymes; however, still resemble each other in that superimposition of one over the other has a substantial overlap, with a [http://en.wikipedia.org/wiki/Root_mean_square_deviation root mean square deviation] (rmsd) of less than 1.76Å between 79% of all <scene name='Sandbox_159/Alphac_1y11/1'>α-carbons</scene><ref name="Linder"/><ref name="Tews"/>. A notable difference in Rv1264 and type II adenylyl cyclase catalytic domains is their relative sizes; Rv1264 has no dimerization arm and several of its loops have been shortened<ref name="Linder"/><ref name="Tews"/>. This results in Rv1264 adenylyl cyclase catalytic domain associating as a dimer with a smaller [http://en.wikipedia.org/wiki/Interface_(chemistry) interface] area than mammal's type II, with interface areas of 1900Å<sup>2</sup> and 3800Å<sup>2</sup>, respectively<ref name="Tews"/>. The active sites the Rv1264 and mammalian type II adenylyl cyclases are even more conserved; the <scene name='Sandbox_159/Activealpha_1y11/2'>α-carbons</scene> in the active site have an rmsd of only 0.69Å, and <scene name='Sandbox_159/Activeatoms_1y11/2'>all atoms</scene> in the active site have a rmsd of 1.17Å<ref name="Linder"/><ref name="Tews"/>.<br />
[[Image:Dimer2.png|frame|left|Rv1264 dimer in its active state.]]<br />
All of the above structural information for the C-terminal catalytic domain is only true when the enzyme is in its active state. The inactive state of the enzyme possesses a disassembled active site, hence no catalytic activity. Relative to the fixed N-terminal domains, each of the C-terminal monomeric domains can transpose up to 6Å and rotate by 55<sup>o</sup><ref name="Tews"/>. This massive change in the C-terminal domains tertiary structure disassembles the active site, moving catalytic residues up to 25Å away from their catalytically active position<ref name="Tews"/>. Not only is the active site disrupted in the C-terminal domain, but the interface area between the two monomers decreases in size from 1900Å<sup>2</sup> to 930Å<sup>2</sup><ref name="Tews"/>.<br />
===== α1-switch =====<br />
The <scene name='Sandbox_159/Alphaoneswitch_1y11/1'>α1-switch</scene> is located within the C-terminal catalytic domain and exists as a compact α-helix when the enzyme is in its active state,<ref name="Tews"/>. Upon inactivation the α-helical conformation of the α1-switch becomes unstable, and it transforms into a random coil<ref name="Tews"/>. Since this switch is in a close proximity to the active site, a major change in structure greatly disrupts the active site, and yields the enzyme inactive<ref name="Tews"/>.<br />
<br />
This structure is conserved in mammalian adenylyl cyclases, where it acts as a contributor for the binding site of the βγ-phosphates of the ATP substrate<ref name="Tesmer3">PMID:11087399</ref>. It also functions in regulation of mammalian adenylyl cyclases, along with another α-helix, it forms the binding site for the G<sub>s</sub>α and G<sub>i</sub>α G protein subunits which regulates the adenylyl cyclases activity<ref name="Zhang"/>.<br />
<br />
==== N-Terminal Regulatory Domain ====<br />
<applet load='1y11' size='380' frame='true' align='right' caption='Rv1264 adenylyl cyclase in its active state.' scene='Sandbox_159/Main_1y11/2'/><br />
<br />
The N-terminal domain functions in regulation; it has a unique mechanism that determines whether the enzyme will be active or not based on the pH of the surrounding solution<ref name="Linder"/>. Each monomer in the catalytically active dimer has ten <scene name='Sandbox_159/Ntermhelix_1y11/1'>α-helices</scene>, when they are dimerized they form a disc like structure<ref name="Tews"/>. A single molecule of <scene name='Sandbox_159/Peg_1y11/1'>pentaethylene glycol</scene> binds in a hydrophobic pocket<ref name="Findeisen">PMID:17482646</ref>. The function of this polyethylene glycol appears to be structural; however, its specific placement and movement between active and inactive forms of the enzyme suggests that it may also function in detecting hydrophobicty changes in the environment<ref name="Findeisen"/>.<br />
===== αN10-switch =====<br />
The <scene name='Sandbox_159/Linkerswitch_1y11/1'>αN10-switch</scene> is located within the linker region and its conformational change has a more drastic effect on the overall enzyme than the α1-switch does<ref name="Tews"/><ref name="Findeisen"/>. When the enzyme is active, the αN10-switch consists of a random coil with a short α-helix; this conformation allows for only a weak interaction between the N-terminal regulatory domain and the C-terminal catalytic domain, allowing the enzyme to exhibit catalytic activity<ref name="Tews"/>. This helix can extend by up to 24Å, which separates the monomeric C-terminal catalytic domains of the dimer<ref name="Tews"/>. This separation of domains lowers their interface area and transposes residues; as discussed above, these changes result in an inactivation of the enzyme, and thus, a major characteristic of the inactive state of the enzyme is an extended αN10-switch<ref name="Tews"/><ref name="Findeisen"/>. As the αN10-switch extends, the <scene name='Sandbox_159/Alpha4_1y11/1'>α4-helix</scene> moves outwards, and the pentaethylene glycol moves into a newly formed cavity by the αN10 helix<ref name="Findeisen"/>. This further supports the idea that the pentaethylene glycol ligand functions not only for structure, but also for regulation<ref name="Findeisen"/>.<br />
<br />
=== Regulation by pH ===<br />
At the beginning of the linker region there is a residue, <scene name='Sandbox_159/His192_1y11/1'>His 192</scene>, which has a considerable influence on regulation by pH<ref name="Tews"/><ref name="Findeisen"/>. At a basic pH the residue has no charge and minimal interactions with the two catalytically important residues <scene name='Sandbox_159/Lysargactive_1y11/2'>Lys 261 (green) and Asp 312 (red)</scene>, which lie 14 Å and 21 Å away from their catalytically active positions<ref name="Tews"/><ref name="Findeisen"/>. The enzyme is inactive at this state, not only because of the Lys 261 and Asp 312 residues, but also because of other major structural components of the catalytic site being disassembled. At an acidic pH Rv1264 becomes active (Optimal pH~5.8)and has up to a 40 fold increase in activity<ref name="Tews"/>. At this acidic pH, His 192 becomes protonated and positively charged; which creates major structural changes throughout the protein, including compaction of both the αN10-switch and α1-switch, contraction of the α4-helix which in turn causes a translocation of the paraethylene glycol ligand<ref name="Linder"/><ref name="Tews"/><ref name="Findeisen"/>. The positively charged His 192 electrostatically repels the Lys 261 and Asp 312 residues which along with other structural changes transposes them 14 Å and 21 Å, respectively, into their catalytically active positions<ref name="Tews"/>.<br />
<br />
<scene name='Sandbox_159/Arg309_1y11/1'>Arginine 309</scene>, a residue which organizes many residues which are important in the C-terminal - N-terminal interaction through hydrogen bonds, is also important for regulation<ref name="Tews"/>. When this residue is mutated, regulation based on pH is lost and the enzyme is constantly active<ref name="Tews"/>.<br />
<br />
Many other residues also contribute to regulation by pH; it is the electrostatic interactions and hydrogen bonds in the C-terminal - N-terminal domain interface which allows Rv1264 adenylyl cyclase to be sensitive to pH<ref name="Tews"/>.<br />
<br />
=== Role ===<br />
''M. tuberculosis'' is a pathogenic bacterium, and thus it faces an array of a host's immune responses to attempt in an attempt to rid of it<ref>PMID:11239406</ref>. One of the hosts defense mechanisms ''M. tuberculosis'' faces is acidification encountered in phagolysosomes. The ability to be able to detect this acidic environment, and have an appropriate response to it may greatly assist ''M. tuberculosis'' infect a host<ref>PMID:15275372</ref><ref>PMID:17342138</ref>. As cAMP levels are increased, acidification of other structures is delayed and elevated cAMP levels activate [http://en.wikipedia.org/wiki/CAMP_receptor_protein cAMP receptor proteins] which in turn regulate transcription<ref>PMID:165421</ref><ref>PMID:15882420</ref>.<br />
<br />
== References ==<br />
<references/></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Adenylyl_cyclase&diff=1077054Adenylyl cyclase2010-04-16T00:35:04Z<p>Travis Eyford: </p>
<hr />
<div>Adenylyl cyclase, also known as adenylate cyclase, is an enzyme which catalyzes the cyclization of [http://en.wikipedia.org/wiki/Adenosine_triphosphate adenosine triphosphate] (ATP) into [http://en.wikipedia.org/wiki/Cyclic_adenosine_monophosphate cyclic adenosine monophosphate] (cAMP) which requires the cleavage of [http://en.wikipedia.org/wiki/Pyrophosphate pyrophosphate] (PPi)<ref name="Taussig">PMID:7814360</ref>.<br />
{{STRUCTURE_1y11| PDB=1y11 | SCENE=Sandbox_159/Main_1y11/1 }}<br />
<br />
== Introduction ==<br />
There are ten isozymes of adenylyl cyclases in mammals, adenylyl cyclase type I-X, (ADCY I-X), and many more in other organisms<ref name="Taussig"/>. All mammalian, and most other adenylyl cyclases belong to class III; most are integral membrane proteins, and all produce cAMP, the ability of which can be activated or inactivated in response to certain conditions or ligands<ref name="Taussig"/>. All mammalian membrane bound adenylyl cyclases are activated by alpha subunits of [http://en.wikipedia.org/wiki/G-protein/ G-proteins], but respond differently to ligands such as magnesium ions, calcium ions, and beta gamma subunits of G proteins<ref name="Taussig"/>. One of the mammalian isozymes, and some prokaryotic forms of adenylyl cyclase respond to environmental conditions, primarily pH<ref>PMID:14512417</ref><ref name="Linder">PMID:11839758</ref>.<br />
<br />
== Reaction ==<br />
=== Reactant ===<br />
The reactant in the reaction catalyzed by adenylyl cyclase is ATP; ATP is the most abundant nucleotide triphosphate in most cells with typical concentrations ranging from 1 to 10mM<ref>PMID:1212224</ref>. This high intracellular concentration allows for cAMP concentrations to rise quickly in response to a specific signal, which is important in many signal transduction and metabolic pathways<ref>PMID:182581</ref>.<br />
<br />
=== Reaction ===<br />
[[Image:CAMP synthesis.png|frame|center]]<br />
The reaction occurs in a single, concerted step, where the oxygen on ATP's 3' hydroxyl group nucleophillically attacks the alpha-phosphate forming a phosphodiester bond and cleaving a pyrophosphate group<ref name="Hurley">PMID:10075642</ref>. In most active sites there is an acidic residue near the 3'OH which functions in its deprotonation, and basic residues by the β-phosphorous to lower the energy of the group for cleavage<ref name="Zhang">PMID:9069282</ref><ref>PMID:14747729</ref>.<br />
<br />
=== Products ===<br />
The main product of this reaction is cAMP, with a side product of PP<sub>i</sub><ref name="Taussig"/>.<br />
==== Cyclic Adenosine Monophosphate ====<br />
In mammals, cAMP acts as a secondary messenger, one of its functions is to control the activity of [http://en.wikipedia.org/wiki/Protein_kinase_A protein kinase A] (PKA)<ref name="Siddappa">PMID:18490653</ref>. In turn, PKA has quite diverse roles in cells, while most of them are associated with metabolism, PKA also plays important roles in transcription, the cell cycle, and [http://en.wikipedia.org/wiki/Apoptosis apoptosis]<ref name="Siddappa"/>. The ultimate fate of cAMP is its transformation into AMP by the cleavage of the phosphodiester bond by 3', 5'-cyclic adenosine monophosphate phosphodiesterase<ref name="Siddappa"/>.<br />
<br />
==== Pyrophosphate ====<br />
Cleavage of the by-product of this reaction, PP<sub>i</sub>, by pyrophosphatase yields two molecules of [http://en.wikipedia.org/wiki/Phosphate inorganic phosphate] (P<sub>i</sub>)<ref>PMID:4964763</ref>. ATP synthase can reincorporate this inorganic phosphate into adenine diphosphate (ADP) to make ATP using energy in the proton motive force<ref>PMID:9242922</ref>.<br />
<br />
== Mammalian Adenylyl Cyclase ==<br />
There are ten isozymes of adenylyl cyclases in mammals, adenylyl cyclase type I-X, (ADCY I-X); In mammals adenylyl cyclase plays an important role in signal transduction pathways in which cAMP is a secondary messenger<ref name="Feinstein">PMID:1719547</ref>.<br />
<br />
ADCY I-IX all share a general structure; They are composed of two trans-membrane regions (M1, M2) which are composed of six membrane-spanning helices and function to keep the enzyme anchored in the membrane, and two cytoplasmic regions (C<sub>1</sub>, C<sub>2</sub>) which can be further sub divided (C<sub>1a</sub>, C<sub>1b</sub>, C<sub>2a</sub>, C<sub>2b</sub>) and are responsible for all catalytic activity, and regulation by G-proteins and [http://en.wikipedia.org/wiki/Forskolin forskolin]<ref name="Feinstein"/>. In solution, the C<sub>1a</sub> and C<sub>2a</sub> domains can form heterodimers with each other, either in the same or different enzymes, or they can form homodimers with their identical units on different enzymes<ref name="Zhang"/>. The C<sub>1b</sub> domain is very large (≈15 kDa) with many regulatory sites, and has a variable structure across isozymes; while the C<sub>2b</sub> domain is nearly non-existent in many isozymes, and has yet to be associated with a particular function<ref name="Zhang"/>. <br />
=== Type II ===<br />
==== Structure ====<br />
<applet load='1ab8' size='300' frame='true' align='right' caption='Type II adenylyl cyclase homodimer with two bound forskolin molecules' scene='Sandbox_159/Full_structure_1ab8/1'/><br />
A monomer of C<sub>2</sub> domain of type II adenylyl cyclase has an internal, hydrophobic, anti-parallel <scene name='Sandbox_159/Beta_sheets_1ab8/1'>β-sheet</scene> surrounded by several, amphipathic <scene name='Sandbox_159/Helix_1ab8/1'>α-helices</scene>, except for an area which needed to form a homodimer with another C<sub>2</sub> domain<ref name="Zhang"/>. Two monomers of C<sub>2</sub> domains of type II adenylyl cyclase bind together in solution to form a <scene name='Sandbox_159/Full_structure_1ab8/1'>Homodimer</scene>, which is necessary for catalytic conversion of ATP to cAMP and PPi<ref name="Hurley"/>. When they are bound they create a deep crevasse spanning the center of their binding site; this crevasse is suited to bind two <scene name='Sandbox_159/Forskolin_1ab8/4'>forskolin</scene> molecules at its ends<ref name="Tesmer">PMID:9417641</ref>. Strong hydrogen bonds are made between oxygen atoms of forskolin and the surrounding peptide backbone, and the rest of the interactions are highly hydrophobic, as the forskolin binding site contains ten aliphatic and aromatic residues<ref name="Tesmer"/>. This binding of forskolin creates a hydrophobic linkage between the monomers, each of which has two different hydrophobic surfaces binding to forskolin; and it is this interaction which makes the homodimer stable<ref name="Zhang"/><ref name="Tesmer"/>. The forskolin also interacts with and properly positions Asn 1025, which is essential for catalytic activity, and it may even interact directly with the ATP<ref name="Tesmer"/>. This homodimer-forskolin complex can be further activated in response to a signal via binding to a G-protein’s βγ-subunit <ref name="Hurley"/><ref name="Tesmer"/>. This βγ-subunit binds to <scene name='Sandbox_159/G-protein_binding_site/1'>residues 956 to 982</scene> which make up part of an α-helix on the outermost layer of the complex<ref name="Hurley"/><ref>PMID:2899356</ref>. <br />
<br />
The <scene name='Sandbox_159/Activesite_1ab8/3'>active site</scene> of this homodimer is located within the crevasse, and is characterized by two highly conserved sets of polar residues (Arg 997 (Green), Asn 1025 (Red), Ser1028(Pink), Arg 1029(Orange), Asp 1031(Yellow), and Ser 1032(Purple))<ref name="Hurley"/><ref name="Zhang"/>. One of these sets is located on each monomeric subunit, on the homodimer they arrange themselves in an anti-parallel fashion, where they point towards each other<ref name="Zhang"/>.<br />
<br />
==== Over-expression Disorders ====<br />
In the brain of mammals, the execution of memory based functions is carried out by the [http://en.wikipedia.org/wiki/Prefrontal_cortex prefrontal cortex] (PFC)<ref name="Wang">PMID:17448997</ref>. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels on neurons close to allow electrochemical signals to flow down the axon and into a synapse; when HCN channels are open, the electropotential signal cannot be transmitted through the cell<ref name="Wang"/>. Exposure of these channels to cAMP causes them open, stopping the transmission of signals, and thus impairs higher cognitive thoughts<ref name="Wang"/>. In patients with schizophrenia, a cAMP regulatory molecule, [http://en.wikipedia.org/wiki/DISC1 Disrupted-in-Schizophrenia 1] (DISC1) is mutated and cannot regulate cAMP levels<ref name="Wang"/>; Thus, elevated cAMP levels may cause schizophrenia. The closing of HCN channels are thought to play a part in other disorders, such as ADHD and bipolar disorder<ref name="Wang"/>. Thus, it is reasonable that regulation of cAMP production by targeting type II adenylyl cyclase, since it is found in the brain, may act as a treatment for these disorders.<br />
<br />
== Rv1264 Adenylyl Cyclase ==<br />
Although adenylyl cyclase is found throughout organisms at a universal level, distantly related organisms have different modifications of the enzyme, each is specialized for a particular task in a particular environment<ref name="Tews">PMID:15890882</ref>. As stated earlier humans have 10 known isozymes of adenylyl cyclase; whereas ''Escherichia coli'' has only one isozyme, and ''Mycobacterium tuberculosis'' has 15<ref name="Tews"/>. One particularly interesting adenylyl cyclase possessed by ''M. tuberculosis'', Rv1264, has a N-terminal which, in a sense, acts as a pH sensor, as it regulates the activity of the enzyme based on the pH of the surrounding solution<ref name="Linder"/><ref name="Tews"/>. This adenylyl cyclase, like most others, belongs to class III, adenylyl cyclases in this class have multiple domains, at least one for catalysis, and another for regulation<ref name="Tews"/>.<br />
=== Structure ===<br />
<applet load='1y11' size='380' frame='true' align='right' caption='Rv1264 adenylyl cyclase monomer in its active state.' scene='Sandbox_159/Main_1y11/2'/><br />
The <scene name='Sandbox_159/Main_1y11/2'>Rv1264</scene> adenylyl cyclase is a 363 residue long protein composed of a catalytic <scene name='Sandbox_159/Cdomain_1y11/1'>C-terminal domain</scene> and a regulatory <scene name='Sandbox_159/Ndomain_1y11/2'>N-terminal domain</scene> which contains a flexible <scene name='Sandbox_159/Connector_1y11/2'>linker region</scene> which connects the two<ref name="Tews"/>. The active structure is a homodimer resembling the mammalian type II homodimer, where an α-helix of one monomer (Chain A) is placed through the central coiled coil of another (Chain B)<ref name="Zhang"/><ref name="Tews"/>. This dimerization places the regulatory domain of chain A in close proximity to the catalytic domain of chain B, and vice versa<ref name="Tews"/>. Two switch elements are present in the protein, one in the C-terminal domain (α1-switch) and another in the linker region (αN10-switch), these allow for large conformational changes to take place in the enzyme in response to relatively small environmental changes<ref name="Linder"/><ref name="Tews"/>. <br />
==== C-Terminal Catalytic Domain ====<br />
The catalytic activity in Rv1264's <scene name='Sandbox_159/Active_1y11/1'>active site</scene> is performed by the residues: Asp 222 (Red), Lys 261 (Blue), Asp 265 (Orange), Arg 298 (Pink), Asp 312 (Yellow), Asn 319 (Purple), Arg 323 (Green)<ref name="Tews"/>. All of these residues create a highly polar environment which is complementary in charge and polarity to the intermediate of the reaction<ref name="Linder"/><ref name="Tews"/>. Residues which guide the phosphates of ATP, arginine 298 and 323, bind a <scene name='Sandbox_159/Sulphate_1y11/1'>sulphate ion</scene> in the active site<ref name="Tesmer2">PMID:10427002</ref>. This sulphate ion is located in the position which will be occupied by the ATP's β-phosphate during catalysis<ref name="Tesmer2"/>. In the process of catalysis the β-phosphate is cleaved from the α-phosphate; this reaction may be made more favourable by lowering the energy though complementary charge associations between the β-phosphate and arginines 298 and 323<ref name="Tews"/><ref name="Tesmer2"/>. Another residue, <scene name='Sandbox_159/Argglycerol_1y11/1'>arginine 296</scene>, binds a <scene name='Sandbox_159/Glycerol_1y11/1'>glycerol</scene> molecule through electrostatic interactions<ref name="Tews"/>. The specific function of this association is unknown, but because of its close proximity to the active site it may play a role in catalysis<ref name="Tews"/>. <br />
<br />
Amino acid sequencing has shown that the sequence between the Rv1264 adenylyl cyclases catalytic domain and mammalian adenylyl cyclases catalytic domain are not well conserved, with only about a 25% correspondence to the mammalian type II adenylyl cyclase discussed above<ref name="Linder"/><ref name="Tews"/>. The two different isozymes; however, still resemble each other in that superimposition of one over the other has a substantial overlap, with a [http://en.wikipedia.org/wiki/Root_mean_square_deviation root mean square deviation] (rmsd) of less than 1.76Å between 79% of all <scene name='Sandbox_159/Alphac_1y11/1'>α-carbons</scene><ref name="Linder"/><ref name="Tews"/>. A notable difference in Rv1264 and type II adenylyl cyclase catalytic domains is their relative sizes; Rv1264 has no dimerization arm and several of its loops have been shortened<ref name="Linder"/><ref name="Tews"/>. This results in Rv1264 adenylyl cyclase catalytic domain associating as a dimer with a smaller [http://en.wikipedia.org/wiki/Interface_(chemistry) interface] area than mammal's type II, with interface areas of 1900Å<sup>2</sup> and 3800Å<sup>2</sup>, respectively<ref name="Tews"/>. The active sites the Rv1264 and mammalian type II adenylyl cyclases are even more conserved; the <scene name='Sandbox_159/Activealpha_1y11/2'>α-carbons</scene> in the active site have an rmsd of only 0.69Å, and <scene name='Sandbox_159/Activeatoms_1y11/2'>all atoms</scene> in the active site have a rmsd of 1.17Å<ref name="Linder"/><ref name="Tews"/>.<br />
[[Image:Dimer2.png|frame|left|Rv1264 dimer in its active state.]]<br />
All of the above structural information for the C-terminal catalytic domain is only true when the enzyme is in its active state. The inactive state of the enzyme possesses a disassembled active site, hence no catalytic activity. Relative to the fixed N-terminal domains, each of the C-terminal monomeric domains can transpose up to 6Å and rotate by 55<sup>o</sup><ref name="Tews"/>. This massive change in the C-terminal domains tertiary structure disassembles the active site, moving catalytic residues up to 25Å away from their catalytically active position<ref name="Tews"/>. Not only is the active site disrupted in the C-terminal domain, but the interface area between the two monomers decreases in size from 1900Å<sup>2</sup> to 930Å<sup>2</sup><ref name="Tews"/>.<br />
===== α1-switch =====<br />
The <scene name='Sandbox_159/Alphaoneswitch_1y11/1'>α1-switch</scene> is located within the C-terminal catalytic domain and exists as a compact α-helix when the enzyme is in its active state,<ref name="Tews"/>. Upon inactivation the α-helical conformation of the α1-switch becomes unstable, and it transforms into a random coil<ref name="Tews"/>. Since this switch is in a close proximity to the active site, a major change in structure greatly disrupts the active site, and yields the enzyme inactive<ref name="Tews"/>.<br />
<br />
This structure is conserved in mammalian adenylyl cyclases, where it acts as a contributor for the binding site of the βγ-phosphates of the ATP substrate<ref name="Tesmer3">PMID:11087399</ref>. It also functions in regulation of mammalian adenylyl cyclases, along with another α-helix, it forms the binding site for the G<sub>s</sub>α and G<sub>i</sub>α G protein subunits which regulates the adenylyl cyclases activity<ref name="Zhang"/>.<br />
<br />
==== N-Terminal Regulatory Domain ====<br />
<applet load='1y11' size='380' frame='true' align='right' caption='Rv1264 adenylyl cyclase in its active state.' scene='Sandbox_159/Main_1y11/2'/><br />
<br />
The N-terminal domain functions in regulation; it has a unique mechanism that determines whether the enzyme will be active or not based on the pH of the surrounding solution<ref name="Linder"/>. Each monomer in the catalytically active dimer has ten <scene name='Sandbox_159/Ntermhelix_1y11/1'>α-helices</scene>, when they are dimerized they form a disc like structure<ref name="Tews"/>. A single molecule of <scene name='Sandbox_159/Peg_1y11/1'>pentaethylene glycol</scene> binds in a hydrophobic pocket<ref name="Findeisen">PMID:17482646</ref>. The function of this polyethylene glycol appears to be structural; however, its specific placement and movement between active and inactive forms of the enzyme suggests that it may also function in detecting hydrophobicty changes in the environment<ref name="Findeisen"/>.<br />
===== αN10-switch =====<br />
The <scene name='Sandbox_159/Linkerswitch_1y11/1'>αN10-switch</scene> is located within the linker region and its conformational change has a more drastic effect on the overall enzyme than the α1-switch does<ref name="Tews"/><ref name="Findeisen"/>. When the enzyme is active, the αN10-switch consists of a random coil with a short α-helix; this conformation allows for only a weak interaction between the N-terminal regulatory domain and the C-terminal catalytic domain, allowing the enzyme to exhibit catalytic activity<ref name="Tews"/>. This helix can extend by up to 24Å, which separates the monomeric C-terminal catalytic domains of the dimer<ref name="Tews"/>. This separation of domains lowers their interface area and transposes residues; as discussed above, these changes result in an inactivation of the enzyme, and thus, a major characteristic of the inactive state of the enzyme is an extended αN10-switch<ref name="Tews"/><ref name="Findeisen"/>. As the αN10-switch extends, the <scene name='Sandbox_159/Alpha4_1y11/1'>α4-helix</scene> moves outwards, and the pentaethylene glycol moves into a newly formed cavity by the αN10 helix<ref name="Findeisen"/>. This further supports the idea that the pentaethylene glycol ligand functions not only for structure, but also for regulation<ref name="Findeisen"/>.<br />
<br />
=== Regulation by pH ===<br />
At the beginning of the linker region there is a residue, <scene name='Sandbox_159/His192_1y11/1'>His 192</scene>, which has a considerable influence on regulation by pH<ref name="Tews"/><ref name="Findeisen"/>. At a basic pH the residue has no charge and minimal interactions with the two catalytically important residues <scene name='Sandbox_159/Lysargactive_1y11/2'>Lys 261 (green) and Asp 312 (red)</scene>, which lie 14 Å and 21 Å away from their catalytically active positions<ref name="Tews"/><ref name="Findeisen"/>. The enzyme is inactive at this state, not only because of the Lys 261 and Asp 312 residues, but also because of other major structural components of the catalytic site being disassembled. At an acidic pH Rv1264 becomes active (Optimal pH~5.8)and has up to a 40 fold increase in activity<ref name="Tews"/>. At this acidic pH, His 192 becomes protonated and positively charged; which creates major structural changes throughout the protein, including compaction of both the αN10-switch and α1-switch, contraction of the α4-helix which in turn causes a translocation of the paraethylene glycol ligand<ref name="Linder"/><ref name="Tews"/><ref name="Findeisen"/>. The positively charged His 192 electrostatically repels the Lys 261 and Asp 312 residues which along with other structural changes transposes them 14 Å and 21 Å, respectively, into their catalytically active positions<ref name="Tews"/>.<br />
<br />
<scene name='Sandbox_159/Arg309_1y11/1'>Arginine 309</scene>, a residue which organizes many residues which are important in the C-terminal - N-terminal interaction through hydrogen bonds, is also important for regulation<ref name="Tews"/>. When this residue is mutated, regulation based on pH is lost and the enzyme is constantly active<ref name="Tews"/>.<br />
<br />
Many other residues also contribute to regulation by pH; it is the electrostatic interactions and hydrogen bonds in the C-terminal - N-terminal domain interface which allows Rv1264 adenylyl cyclase to be sensitive to pH<ref name="Tews"/>.<br />
<br />
=== Role ===<br />
''M. tuberculosis'' is a pathogenic bacterium, and thus it faces an array of a host's immune responses to attempt in an attempt to rid of it<ref>PMID:11239406</ref>. One of the hosts defense mechanisms ''M. tuberculosis'' faces is acidification encountered in phagolysosomes. The ability to be able to detect this acidic environment, and have an appropriate response to it may greatly assist ''M. tuberculosis'' infect a host<ref>PMID:15275372</ref><ref>PMID:17342138</ref>. As cAMP levels are increased, acidification of other structures is delayed and elevated cAMP levels activate [http://en.wikipedia.org/wiki/CAMP_receptor_protein cAMP receptor proteins] which in turn regulate transcription<ref>PMID:165421</ref><ref>PMID:15882420</ref>.<br />
<br />
== References ==<br />
<references/></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Adenylyl_cyclase&diff=1077053Adenylyl cyclase2010-04-16T00:33:50Z<p>Travis Eyford: New page: ==This is a placeholder== This is a placeholder text to help you get started in placing a Jmol applet on your page. At any time, click "Show Preview" at the bottom of this page to see how...</p>
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{{STRUCTURE_3cin | PDB=3cin | SCENE= }}</div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062068Sandbox 1542010-03-29T04:34:02Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref name="Graceffa">PMID:12813032</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref name="Holmes1">PMID:19158779</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref name="Holmes2">PMID:2395461</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref name="Pfaendtner">PMID:19620726</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref name="Mitchinson">PMID:1493331</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref name="Chen">PMID:10637608</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref name="Carlier">PMID:3801442</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å<ref name="Holmes2"/>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref name="Pfaendtner"/>A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="Graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="Pfaendtner"/><ref name="Graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="Graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker">PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref name="Mitchinson"/>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref name="Chen"/>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref name="Holmes2"/><ref name="Holmes3>PMID:14508495</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref name="Mitchinson"/>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062067Sandbox 1542010-03-29T04:32:05Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref name="Graceffa">PMID:12813032</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref name="Holmes1">PMID:19158779</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref name="Holmes2">PMID:2395461</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref name="Pfaendtner">PMID:19620726</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref name="Mitchinson">PMID:1493331</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref name="Chen">PMID:10637608</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref name="Carlier">PMID:3801442</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å<ref name="Holmes2"/>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref name="Pfaendtner"/>A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="Graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="pfaendter"/><ref name="Graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="Graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker">PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref name="Mitchinson"/>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref name="Chen"/>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref name="Holmes2"/><ref name="Holmes3>PMID:14508495</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref name="Mitchinson"/>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062066Sandbox 1542010-03-29T04:30:57Z<p>Travis Eyford: /* Actin-Myosin */</p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref name="Graceffa">PMID:12813032</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref name="Holmes1">PMID:19158779</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref name="Holmes2">PMID:2395461</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref name="Pfaendtner>PMID:19620726</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref name="Mitchinson">PMID:1493331</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref name="Chen">PMID:10637608</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref name="Carlier">PMID:3801442</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å<ref name="Holmes2"/>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref name="Pfaendtner"/>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="Graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="pfaendter"/><ref name="Graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="Graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker">PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref name="Mitchinson"/>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref name="Chen"/>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref name="Holmes2"/><ref name="Holmes3>PMID:14508495</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref name="Mitchinson"/>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062065Sandbox 1542010-03-29T04:29:41Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref name="Graceffa">PMID:12813032</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref name="Holmes1">PMID:19158779</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref name="Holmes2">PMID:2395461</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref name="Pfaendtner>PMID:19620726</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref name="Mitchinson">PMID:1493331</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref name="Chen">PMID:10637608</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref name="Carlier">PMID:3801442</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å<ref name="Holmes2"/>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref name="Pfaendtner"/>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="Graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="pfaendter"/><ref name="Graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="Graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker">PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref name="Mitchinson"/>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref name="Chen"/>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref name="Holmes"2/><ref name="Holmes3>PMID:14508495</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref name="Mitchinson"/>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062064Sandbox 1542010-03-29T04:28:02Z<p>Travis Eyford: /* Polymer */</p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref name="Graceffa">PMID:12813032</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref name="Holmes1">PMID:19158779</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref name="Holmes2">PMID:2395461</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref name="Pfaendtner>PMID:19620726</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref name="Mitchinson">PMID:1493331</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref name="Chen">PMID:10637608</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref name="Carlier">PMID:3801442</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å<ref name="Holmes2"/>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref name="Pfaendtner">. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="Graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="pfaendter"/><ref name="Graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="Graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker">PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref name="Mitchinson"/>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref name="Chen"/>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref name="Holmes"2/><ref name="Holmes3>PMID:14508495</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref name="Mitchinson"/>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062063Sandbox 1542010-03-29T04:26:33Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref name="Graceffa">PMID:12813032</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref name="Holmes1">PMID:19158779</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref name="Holmes2">PMID:2395461</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref name="Pfaendtner>PMID:19620726</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref name="Mitchinson">PMID:1493331</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref name="Chen">PMID:10637608</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref name="Carlier">PMID:3801442</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å<ref name="Holmes2"/>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref>pfaendtner</ref>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="Graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="pfaendter"/><ref name="Graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="Graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker">PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref name="Mitchinson"/>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref name="Chen"/>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref name="Holmes"2/><ref name="Holmes3>PMID:14508495</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref name="Mitchinson"/>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062062Sandbox 1542010-03-29T04:21:06Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref name="Graceffa">PMID:12813032</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref name="Holmes1">PMID:19158779</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref name="Holmes2">PMID:2395461</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref name="Pfaendtner>PMID:19620726</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref name="Mitchinson">PMID:1493331</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref name="Chen">PMID:10637608</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref name="Carlier">PMID:3801442</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å<ref name="Holmes2"/>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref>pfaendtner</ref>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="Graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="pfaendter"/><ref name="Graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="Graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker">PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref name="Mitchinson">. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref name="Chen">. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref name="Holmes"2><ref name="Holmes3>PMID:14508495</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref name="Mitchinson">.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062056Sandbox 1542010-03-29T04:04:39Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref>PMID:12813032</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref>Holmes1</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref>Holmes 2 </ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref>pfaendtner</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref>mitchinson</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref>chen</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref>clasier</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å.<ref name="holmes">PMID:2395461</ref>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref>pfaendtner</ref>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="pfaendter"/><ref name="graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker">PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref>mitchinson</ref>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref>chen</ref>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref> Holmes 2, Holmes et al 3</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref>Mitchinson</ref>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062055Sandbox 1542010-03-29T03:59:41Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref>Graceffa</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref>Holmes1</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref>Holmes 2 </ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref>pfaendtner</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref>mitchinson</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref>chen</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref>clasier</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å.<ref name="holmes">PMID:2395461</ref>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref>pfaendtner</ref>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="pfaendter"/><ref name="graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker">PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref>mitchinson</ref>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref>chen</ref>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref> Holmes 2, Holmes et al 3</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref>Mitchinson</ref>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1062054Sandbox 1542010-03-29T03:58:54Z<p>Travis Eyford: </p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and P<sub>i</sub> and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref>Graceffa</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref>Holmes1</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref>Holmes 2 </ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. Also, F-actin possesses ATPase function where it is minimal in G-actin. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref>pfaendtner</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref>mitchinson</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref>chen</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref>clasier</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
== Structure == <br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, the "Holmes model" was accepted. In contrast, over the G-actin structure has been determined independently over 30 times. A higher resolution F-actin model was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref name="oda">PMID:19158791</ref>.<br />
<br />
=== F-actin Monomer and Polymer ===<br />
==== Monomer ====<br />
[[image:2zwh_domain_colours2.png|thumb|Domains of F-actin monomer|frame|left]]<br />
==== Polymer ==== <br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å.<ref name="holmes">PMID:2395461</ref>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref name="oda" />. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref>pfaendtner</ref>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref name="oda" />.<br />
=== Nucleotide-State-Dependent Conformational Changes ===<br />
The state of the bound phosphorylated nucleotide affects what conformation the F-actin monomer undertakes. The presence of a gamma-phosphate in the active site causes the rotation of a Ser14 residue. This change leads to HIC73 (4-methyl histidine) becoming shifted, which alters the F-actin active site and causes a conformational change in the D-loop. The HIC73 is located in the sensor loop, or the "switch" for linking changes in bound nucleotide to conformational changes<ref name="graceffa"/>. In ATP-actin and ADP-P<sub>i</sub>-actin, the D-loop is unstructured. In the ADP-bound form of F-actin, an alpha helix is commonly apparent in the D-loop of the monomer<ref name="pfaendter"/><ref name="graceffa"/>. Although the alpha-helix is not observed in this Oda model of F-actin, it is acknowkledged by Oda et. al that the experimental results could have lead to an extended alpha-helix in the model<ref name="oda"/>. <br />
==== Domains ====<br />
<applet load='2zwh' size='275' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
The structure of a single unit of F-actin arises from one polypeptide chain with two domains, as observed by the figure on the left. The nucleotide binding cleft, site of ATP hydrolysis, can be observed between the two domains. Movement of the domains allows for the open and closed F-actin conformations. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> peptide bonds of residues 141-142 and 335-336</scene>, shown in purple. According to Oda et al., during the transition from G- to F- actin, Domain 2 is believed to tilt 20&deg; and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis<ref name="oda" />.<br />
<br />
==== Stability ====<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the F-actin complex is achieved by a series of <scene name='Sandbox_154/2zwh_saltbridge/1'>salt bridge</scene> formations involving arginine 206, 183, 177 (purple); glutamate 72(blue), aspartate 187(green), 179 and 4-methyl histidine 73(yellow). Additional stability is believed to arise from a break in the interaction between residues <scene name='Sandbox_154/2zwh_leu_val/2'>108-111 and Val165 and Ile175</scene> in the same half of their respective domains to a new interaction between <scene name='Sandbox_154/2zwh_leu_thr/2'>Leu110 and Thr194</scene> where a much greater distance is observed between them<ref name="oda" />.<br />
<br />
==== Active Site ====<br />
Upon actin binding on the plus end of the actin filament, the ATPase function is activated. The conformational change from G- to F- actin promotes the catalytic activity because of the 20&deg; shift leading to a more closed binding site; this conformational change is stabilized also by the diagonal subdomain interaction between Leu110 and Thr194. <br />
As a result of these conformational changes, the Gln137 residue of actin is moved closer to the ATP-Ca<sup>2+</sup> ligand. Gln137 holds a water molecule, and placing it in close proximity to ATP allows for the gamma-phosphate to become cleaved. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref name="graceffa"/>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily exclusive of one another. <br />
<br />
<br />
The dynamic functions of f-actin are heavily involved with [http://en.wikipedia.org/wiki/Cell_migration/ cell migration]<ref name="stricker"><ref>PMID:19913792</ref>.<br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref>mitchinson</ref>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref>chen</ref>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref> Holmes 2, Holmes et al 3</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref>Mitchinson</ref>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1061573Sandbox 1542010-03-26T23:23:55Z<p>Travis Eyford: /* Actin-Myosin */</p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and Pi and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref>Graceffa</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref>Holmes1</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref>holmes2</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref>pfaendtner</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref>mitchinson</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref>chen</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref>clasier</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
== Structure == <br />
<applet load='2zwh' size='222' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
[[image:2zwh_domain_colours2.png|frame|left]]<br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, despite the importance of F-actin in eukaryotic cells, this speculated structure was accepted. A higher resolution structure was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref> Oda T, Iwasa M, Aihara T, Maéda Y, and Narita A. 2009. The nature of the globular-to fibrous actin transition. Nature,457(7228):441-445. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/19158791/ 19158791]</ref>.<br />
=== Domains of F-actin Unit ===<br />
Structure of a unit of F-actin with domains from a single polypeptide chain. Note the cleft between the two domains houses the nucleotide phosphate ligand and the Ca2+ metal ion ligand. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> 141-142 and 335-336 peptide bonds</scene>, shown in purple. According to Oda et al.<ref>oda</ref>Domain 2 is believed to tilt 20 degrees and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis. <br />
=== Stability ===<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the f-actin complex is achieved by a series of salt bridge formations involving arginine, glutamate, aspartate, and HIC73, a charged methylated histidine residue. Additional stability is believed to arise from interactions across subunits with residues Leu110 and Thr194<ref>oda</ref>. <br />
<br />
<br />
=== Active Site ===<br />
The cleavage of the gamma-phosphoryl from the bound ATP is a result of a conformational change upon binding that moves the Gln137 residue closer to the ATP-Ca2+ ligand. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref>graceffa</ref>.<br />
<br />
=== Polymer F-actin ===<br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å. <ref> Holmes, K.C., Popp, D., Gebhard, W. and Kabsch, W. 1990. Atomic model of the actin filament. Nature,347(6288):44-49. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/2395461/ 2395461]</ref>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref>oda</ref>. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref>pfaendtner</ref>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref>oda</ref>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily distinct. <br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref>mitchinson</ref>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref>chen</ref>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin allows myosin to preferentially bind F-actin over G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref> Holmes 2, Holmes et al 3</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref>Mitchinson</ref>.<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1061566Sandbox 1542010-03-26T23:16:58Z<p>Travis Eyford: /* Assembly */</p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and Pi and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref>Graceffa</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref>Holmes1</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref>holmes2</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which polymerizes to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>calcium ion</scene> ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref>pfaendtner</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref>mitchinson</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP and Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref>chen</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref>clasier</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
== Structure == <br />
<applet load='2zwh' size='222' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
[[image:2zwh_domain_colours2.png|frame|left]]<br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, despite the importance of F-actin in eukaryotic cells, this speculated structure was accepted. A higher resolution structure was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref> Oda T, Iwasa M, Aihara T, Maéda Y, and Narita A. 2009. The nature of the globular-to fibrous actin transition. Nature,457(7228):441-445. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/19158791/ 19158791]</ref>.<br />
=== Domains of F-actin Unit ===<br />
Structure of a unit of F-actin with domains from a single polypeptide chain. Note the cleft between the two domains houses the nucleotide phosphate ligand and the Ca2+ metal ion ligand. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> 141-142 and 335-336 peptide bonds</scene>, shown in purple. According to Oda et al.<ref>oda</ref>Domain 2 is believed to tilt 20 degrees and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis. <br />
=== Stability ===<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the f-actin complex is achieved by a series of salt bridge formations involving arginine, glutamate, aspartate, and HIC73, a charged methylated histidine residue. Additional stability is believed to arise from interactions across subunits with residues Leu110 and Thr194<ref>oda</ref>. <br />
<br />
<br />
=== Active Site ===<br />
The cleavage of the gamma-phosphoryl from the bound ATP is a result of a conformational change upon binding that moves the Gln137 residue closer to the ATP-Ca2+ ligand. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref>graceffa</ref>.<br />
<br />
=== Polymer F-actin ===<br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å. <ref> Holmes, K.C., Popp, D., Gebhard, W. and Kabsch, W. 1990. Atomic model of the actin filament. Nature,347(6288):44-49. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/2395461/ 2395461]</ref>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref>oda</ref>. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref>pfaendtner</ref>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref>oda</ref>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily distinct. <br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref>mitchinson</ref>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref>chen</ref>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin means that myosin preferentially binds to F-actin and not G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref> Holmes 2, Holmes et al 3</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref>Mitchinson</ref>. <br />
<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1061563Sandbox 1542010-03-26T23:05:51Z<p>Travis Eyford: /* Introduction */</p>
<hr />
<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
<br />
== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP to ADP and Pi and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref>Graceffa</ref>. Because of the similarity observed in ''Escherichia Coli'''s, Hsc70 and ATPase domain of actin, it is believed that the two proteins have a common ancestory<ref>Holmes1</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of possible common ancestory<ref>holmes2</ref>. <br />
<br />
Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
<br />
== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which transitions to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>Ca2+</scene> ion ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
<br />
<br />
<br />
Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G- and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref>pfaendtner</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref>mitchinson</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP+Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref>chen</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref>clasier</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
<br />
<br />
<br />
== Structure == <br />
<applet load='2zwh' size='222' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
[[image:2zwh_domain_colours2.png|frame|left]]<br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, despite the importance of F-actin in eukaryotic cells, this speculated structure was accepted. A higher resolution structure was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref> Oda T, Iwasa M, Aihara T, Maéda Y, and Narita A. 2009. The nature of the globular-to fibrous actin transition. Nature,457(7228):441-445. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/19158791/ 19158791]</ref>.<br />
=== Domains of F-actin Unit ===<br />
Structure of a unit of F-actin with domains from a single polypeptide chain. Note the cleft between the two domains houses the nucleotide phosphate ligand and the Ca2+ metal ion ligand. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> 141-142 and 335-336 peptide bonds</scene>, shown in purple. According to Oda et al.<ref>oda</ref>Domain 2 is believed to tilt 20 degrees and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis. <br />
=== Stability ===<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the f-actin complex is achieved by a series of salt bridge formations involving arginine, glutamate, aspartate, and HIC73, a charged methylated histidine residue. Additional stability is believed to arise from interactions across subunits with residues Leu110 and Thr194<ref>oda</ref>. <br />
<br />
<br />
=== Active Site ===<br />
The cleavage of the gamma-phosphoryl from the bound ATP is a result of a conformational change upon binding that moves the Gln137 residue closer to the ATP-Ca2+ ligand. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref>graceffa</ref>.<br />
<br />
=== Polymer F-actin ===<br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å. <ref> Holmes, K.C., Popp, D., Gebhard, W. and Kabsch, W. 1990. Atomic model of the actin filament. Nature,347(6288):44-49. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/2395461/ 2395461]</ref>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref>oda</ref>. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref>pfaendtner</ref>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref>oda</ref>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily distinct. <br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref>mitchinson</ref>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref>chen</ref>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin means that myosin preferentially binds to F-actin and not G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref> Holmes 2, Holmes et al 3</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref>Mitchinson</ref>. <br />
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==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=Sandbox_154&diff=1061561Sandbox 1542010-03-26T23:01:05Z<p>Travis Eyford: /* F-Actin */</p>
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<div>{{STRUCTURE_2zwh| PDB=2zwh | SCENE=Sandbox_154/Black_background/1 }} <br />
= F-Actin = <br />
'''Filamentous actin''' ('''F-actin''') units are also referred to as [http://en.wikipedia.org/wiki/microfilaments/ microfilament] <ref> Microfilaments - Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/Microfilaments. Date accessed: March 16th, 2010. </ref> and are highly conserved, proteinous components found near ubiquitously in eukaryotic cytoskeletons. F-actin and other [http://en.wikipedia.org/wiki/actin/ actin] proteins generally have structural roles in cells. <br />
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== Introduction ==<br />
Actin is found in nearly all eukaryotic cells and is known primarily for its function as a structural and translocation protein. It also has an ATPase function, as it hydrolyzes ATP --> ADP + Pi and undergoes conformational changes with each hydrolysis. Actin belongs to the actin superfamily, which includes other proteins such as Hsp70(DnaK), Hsc70, and hexokinase, because of its nucelotide-dependent conformational change<ref>Graceffa</ref>. Because of the similarity observed in ''E.Coli'' Hsc70 and the ATPase domain of actin, it is believed there was a common ancestor between the two proteins<ref>Holmes1</ref>. Prokaryotes are not known to have actin, but do however have an actin homologue, MreB, which also leads to the idea of a possible common ancestor<ref>holmes2</ref>. <br />
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Actin occurs in two forms: globular actin (G-actin), the free monomeric units of actin, and filamentous actin (F-actin) which is the polymer form. These two forms exist in a dynamic equilibrium with one another as ATP-associated polymerization and depolymerization occur continuously within the cell.<br />
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== Assembly == <br />
<applet load='1j6z' size='200' color='black' frame='true' align='left' caption='Globular Actin (G-actin): PDB identifier [http://www.rcsb.org/pdb/explore/explore.do?structureId=1J6Z/ 1J6Z].' scene='Sandbox_154/1j6z_black/2'/><br />
'''G-actin''' is the free monomeric form of actin which transitions to F-actin. The structures of globular and filamentous actin are distinct from one another in numerous ways, despite the fact that G-actin comprises F-actin. When the monomeric actin becomes polymerized into F-actin, the unit becomes flattened. G-actin appears to have more <scene name='Sandbox_154/1j6z_calcium/1'>Ca2+</scene> ion ligands in its structure, and also has the ligand RHO as opposed to 4-methyl histidine as found in the F-actin structure. <br />
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<br />
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Formation of F-actin is a dynamic process of assembly and disassembly which has been termed “treadmilling”. <br />
The transition between G- and F-actin begins with a stabilized oligomer of ATP-actin units formed through a nucleation-condensation type fold pattern<ref>pfaendtner</ref>. Addition of ATP-monomeric units to either end subsequently occurs, however, because of a difference in charge polarity in the two ends, there is preferential addition to what is termed the "plus (+) end" or the "barbed-end". On the opposite end, the "minus (-) end" or the "pointed end", there is preferential dissociation of actin units<ref>mitchinson</ref>. After attachment of the ATP-bound actin, hydrolysis of the ATP occurs yielding the ADP+Pi bound state. Subsequent loss of a Pi leaves the ADP-actin state<ref>chen</ref>. Because of the potential for addition or removal of monomeric units to occur at both ends, the assembly of F-actin may be described in terms of equilibrium. However, because the rate of ATP-actin association is ten-fold that of ADP-actin dissociation, the f-actin has the appearance of moving forward, or "treadmilling"<ref>clasier</ref>. ADP-actin monomers dissociate at the minus end and become recycled to ATP-actin so polymerization at the plus end may occur once again.<br />
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== Structure == <br />
<applet load='2zwh' size='222' color='black' frame='true' align='right' caption='Filamentous Actin (F-actin)' scene='Sandbox_154/2zwh_black_domains/1'/><br />
[[image:2zwh_domain_colours2.png|frame|left]]<br />
=== History of the structure ===<br />
The F-actin protein was discovered by Straub in 1942. The structure was speculated based on a low-resolution x-ray crystallograph found in 1990 by Holmes et al. and over this time, despite the importance of F-actin in eukaryotic cells, this speculated structure was accepted. A higher resolution structure was only recently deposited in the PDB databank in Decemeber 2008 by Oda et al. <ref> Oda T, Iwasa M, Aihara T, Maéda Y, and Narita A. 2009. The nature of the globular-to fibrous actin transition. Nature,457(7228):441-445. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/19158791/ 19158791]</ref>.<br />
=== Domains of F-actin Unit ===<br />
Structure of a unit of F-actin with domains from a single polypeptide chain. Note the cleft between the two domains houses the nucleotide phosphate ligand and the Ca2+ metal ion ligand. <br />
Domain movement is made possible by rotation about the <scene name='Sandbox_154/2zwh_helix_domains_2/1'> 141-142 and 335-336 peptide bonds</scene>, shown in purple. According to Oda et al.<ref>oda</ref>Domain 2 is believed to tilt 20 degrees and fit itself with Domain 1, thus giving a flatter conformation than the free G-actin. It is not certain whether this flattening occurs before or after ATP hydrolysis. <br />
=== Stability ===<br />
The flattened folded form of F-actin requires different stabilization mechanisms than the free monomeric G-actin form. Stability of the f-actin complex is achieved by a series of salt bridge formations involving arginine, glutamate, aspartate, and HIC73, a charged methylated histidine residue. Additional stability is believed to arise from interactions across subunits with residues Leu110 and Thr194<ref>oda</ref>. <br />
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<br />
=== Active Site ===<br />
The cleavage of the gamma-phosphoryl from the bound ATP is a result of a conformational change upon binding that moves the Gln137 residue closer to the ATP-Ca2+ ligand. Release of the inorganic phosphate occurs via the conformational change of the flexible "D-loop" into an ordered alpha-helix<ref>graceffa</ref>.<br />
<br />
=== Polymer F-actin ===<br />
F-actin has the appearance of two right-handed helices, with a gradual twist around one another. It is actually composed of repeats of 13 actin units for every 6 left-handed turns, spanning a length of 350 Å. <ref> Holmes, K.C., Popp, D., Gebhard, W. and Kabsch, W. 1990. Atomic model of the actin filament. Nature,347(6288):44-49. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/2395461/ 2395461]</ref>. Including the ADP and Ca<sup>2+</sup>, the F-actin molecule as shown here consists of 377 residues (43kDa), two major domains separated by a nucleotide-binding cleft<ref>oda</ref>. Depending on the state of the bound nucleotide, the most stable conformation of F-actin changes. In its ATP and ADP + Pi nucleotide bound states, it has a closed binding cleft. In its ADP only bound state, it has a wider binding cleft<ref>pfaendtner</ref>. A characteristic trait of actin is that the domains remain twisted relative to one another, despite the nucleotide-state-dependent conformational changes<ref>oda</ref>.<br />
<br />
== Function == <br />
F-actin performs a structural, mechanical, and enzymatic role within eukaryotic cells. These functions are not necessarily distinct. <br />
<br />
==== Cytoskeleton ==== <br />
F-actin is the most abundant component of the cytoskeleton of eukaryotes. It provides large amounts of tensile strength, considering its thin size. In cases where the flexibility is not desirable as a strucutral component, crosslinkages can be formed between F-actin polymers to give greater stiffness and support<ref>mitchinson</ref>. <br />
<br />
Elongation of F-actin branches leads to the phenomenon of pushing of the plasma membrane forward in lamellopodial and filopodial extension<ref>chen</ref>. This process relies on the dynamic equlibrium state in which G- and F-actin exist, as it is the continual polymerization of actin units on the leading edge that propels the membrane extension. Without the enzymatic ATPase function of F-actin, this process would not be possible. <br />
<br />
==== Actin-Myosin ==== <br />
The relatively flatter shape of F-actin as compared to G-actin means that myosin preferentially binds to F-actin and not G-actin. This means that F-actin is the functional form of actin composing a large part of the thin filaments that function in muscle contraction<ref> Holmes 2, Holmes et al 3</ref>. Additionally, the structure of F-actin gives it large resistance to extensive forces, such as those experienced in muscle contraction<ref>Mitchinson</ref>. <br />
<br />
<br />
==References==<br />
<references/><br />
<br />
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr></div>Travis Eyfordhttps://proteopedia.org/index.php?title=File:Dimer2.png&diff=1061488File:Dimer2.png2010-03-26T20:11:16Z<p>Travis Eyford: </p>
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<div></div>Travis Eyfordhttps://proteopedia.org/index.php?title=File:CAMP_synthesis.png&diff=1061306File:CAMP synthesis.png2010-03-26T09:04:59Z<p>Travis Eyford: cAMP and PPi formation from ATP cayalyzed by adenylyl cyclase</p>
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<div>cAMP and PPi formation from ATP cayalyzed by adenylyl cyclase</div>Travis Eyfordhttps://proteopedia.org/index.php?title=File:Full_structure.pdb&diff=1060689File:Full structure.pdb2010-03-24T22:26:55Z<p>Travis Eyford: full structure- 1ab8</p>
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<div>full structure- 1ab8</div>Travis Eyfordhttps://proteopedia.org/index.php?title=File:Rxn.png&diff=1058090File:Rxn.png2010-03-19T04:14:58Z<p>Travis Eyford: Reaction catalyzed by adenylyl cyclase.</p>
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<div>Reaction catalyzed by adenylyl cyclase.</div>Travis Eyford