Proteopedia - User contributions [en]

Calmodulin

2010-10-26T09:40:12Z

Enrico Ravera:

[[Image:1prw.png|left|200px|thumb|Crystal structure of bovine brain Ca++ calmodulin in a compact form, [[1prw]]]]
{{STRUCTURE_1prw| PDB=1prw | SIZE=300| SCENE=Calmodulin/Cv/1 |right|CAPTION=Calmodulin, [[1prw]] }}

[[Calmodulin]] (CaM) – calcium modulated protein – regulates various protein targets. It is used by various proteins as calcium sensor and signal transducer by binding to their calcium binding domain (CBD). It undergoes conformational change upon binding Ca++ via its 4 EF hand motives and can undergo post-translational modification.
The images at the left and at the right correspond to one representative calmodulin, ''i.e.'' crystal structure of bovine brain Ca++ calmodulin in a compact form ([[1prw]]).

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== [[Maximum Occurrence]] of Calmodulin Conformations ==

[[Maximum Occurrence]], a method for making rigorous numerical assessments about the maximum percent of time that a conformer of a flexible macromolecule can exist and still be compatible with the experimental data, was used to probe the conformational disorder of Calmodulin<ref>doi:10.1021/ja1063923</ref>. 
[[Image:Movie_MOforproteopedia.gif|thumb|center|400px|Figure 3: Orientation tensor representation for 400 conformational states of Calmodulin, color coded according to their MO values (from less than 5% in blue to more than 30% in red).To better explain their meaning, 10 randomly chosen models are shown as cartoons and then replaced by the three axes of their color-coded orientation tensors.]] 
It was shown that the open ([[1cll]]) and closed ([[1prw]]) conformers can have MO of only 15% and 5% respectively.

== Calmodulin in Motion ==

The clip represents Calmodulin in motion. At the beginning it is shown moving in the unbound form (ApoCaM), and it changes its conformation when Calcium ions are present in the medium (CaCaM).

<center><swf width="720" height="576">/flash/Apo_CaM_CaCaM.swf</swf></center>

Motion of ApoCaM is elaborated on the basis of 23 conformations derived from NMR file [[1cfc]], using the 3D animation program Blender, and according to a system to be published soon (Zini et al., manuscript in preparation). The transition from ApoCaM to CaCaM is elaborated with Blender starting with conformation 21 of 1cfc to arrive in conformation 11 of pdb file 1x02.

Surface rendering is also elaborated using Blender, and shows the lipophilic potential as a scale of white-black and smooth-rough, form the most lipophilic to the hydrophilic. Electrostatic potential is represented as a series of lines moving in the direction Positive to Negative, elaborated according to a scheme to be published soon (Andrei et al., in preparation). As most lines are moving towards Calmodulin, one can learn that the protein is slightly acidic (negative partial charges on its surface).

This movie was created by Andrei, Zini et al., of the
[http://www.scivis.ifc.cnr.it Scientific Visualization Unit],
Institute of Clinical Physiology - CNR of Itlay.

[http://www.molmovdb.org/cgi-bin/morph.cgi?ID=b097743-28520 Conformational change of Calmodulin]

<applet load='1cll' size='350' color='white' frame='true' align='right' caption='Calmodulin' scene='Calmodulin/Inicio/1' />

<scene name='Calmodulin/Inicio/1'>Unbound</scene>,
<scene name='Calmodulin/Bound/4'>Bound</scene>

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== 3D Structures of Calmodulin ==

=== Native CaM ===

[[1prw]], [[1deg]] – bCaM - bovine 
[[1up5]] – cCaM – chicken 
[[1clm]], [[1osa]], [[1exr]] – PtCaM - ''Paramecium tetraurelia'' 
[[3cln]] – rCaM - rat 
[[1x02]], [[1dmo]] – XlCaM – NMR - ''Xenopus laevis'' 
[[2k61]], [[2k0e]] – hCaM – NMR - human 
[[1y6w]], [[1cll]] - hCaM 
[[4cln]] – DmCaM - ''Drosophila melanogaster'' 
[[1rfj]] – CaM – potato 
[[1ooj]] – CaM – ''Caenorhabditis elegans'' 

=== Mutant CaM ===

[[1ahr]] – cCaM (mutant) 
[[2k0j]], [[1sw8]] – hCaM (mutant) – NMR 

=== apo CaM ===

[[1lkj]] – yapoCaM – NMR -yeast 
[[1cfc]], [[1cfd]] - XlapoCaM – NMR 
[[1qx5]] – rapoCaM – rat 

=== CaM N-terminal ===

[[2i08]] - hCaM N-terminal (mutant) 
[[1j7o]] - hCaM N-terminal – NMR 
[[1f70]] - XlCaM N-terminal – NMR 
[[2ro8]], [[2roa]] - sCaM N-terminal+Ca – NMR - soybean 
[[2ro9]], [[2rob]] - sCaM C-terminal+Ca – NMR 
[[1j7o]], [[1j7p]] - hCaM N-terminal – NMR 
[[3ifk]], [[3b32]] – rCaM N-terminal 
[[1f54]], [[1f55]] – yapoCaM N-terminal – NMR 

=== CaM C-terminal ===

[[1f71]] - XlCaM C-terminal – NMR 
[[1cmg]] – bCaM C-terminal – NMR 
[[1fw4]] - bCaM C-terminal 
[[1cmf]] – bapoCaM C-terminal – NMR 
[[2hf5]] - hCaM EF2 EF3 – NMR 

=== CaM+ cations (not calcium) ===

[[2ksz]] – sCaM N-terminal+Mg – NMR 
[[1ak8]] - bCaM N-terminal+Ce – NMR 
[[2eqc]] – XlCaM C-terminal+Mg– NMR 
[[2pq3]] – rCaM+Zn 
[[2v01]] – hCaM+Pb 
[[2v02]] – hCaM+Ba 
[[1n0y]] – PtCaM+Pb 

=== CaM small molecule complexes ===

[[3if7]] – bCaM+sphingosylphosphorylcholine 
[[1qiv]], [[1qiw]] – bCaM+DPDv
[[1a29]], [[1lin]] – bCaM+trifluoperazine 
[[1ctr]] - hCaM+trifluoperazine 
[[2kug]], [[2kuh]] - hCaM N-terminal EF1 EF2+halothane – NMR 
[[2kdu]] – XlCaM+MUNC13-1 – NMR 
[[1mux]] – XlCaM+W-7 – NMR 

=== CaM complexed with protein CBD domains ===

[[3gp2]] – cCaM+CaM kinase II δ chain 
[[3gof]], [[2o60]] – cCaM+nitric oxide synthase CBD 
[[1niw]] - rCaM+nitric oxide synthase CBD 
[[3hr4]] - hCaM+nitric oxide synthase CBD 
[[2x0g]], [[1yr5]], [[1wrz]] – hCaM+death-associated protein kinase 1 (DAP) 
[[2kne]] – hCaM+PMCA C-terminal CBD 
[[3ewt]], [[3ewv]] – hCaM+TNFR fragment 
[[2kn2]] – sCaM C-terminal+NtMKP1 CBD – NMR 
[[2w73]], [[2jzi]], [[2r28]] – hCaM+Ser/Thr phosphatase CBD 
[[3bya]] – hCaM+glutamate receptor peptide 
[[2hqw]] - rCaM+glutamate receptor peptide 
[[2k3s]] – CaM+smoothelin-like protein 1 – NMR 
[[3g43]], [[2vay]], [[2f3y]], [[2f3z]], [[2be6]] – hCaM+calcium channel CAV1.2 
[[3bxk]], [[3bxl]] – rCaM+ calcium channel peptide 
[[1cff]] - XlCaM+ calcium channel CBD - NMR 
[[1g4y]] - rCaM+ potassium channel CBD 
[[1qx7]] – rapoCaM+potassium channel peptide 
[[2o5g]] – cCaM+ myosin light chain kinase peptide 
[[2ix7]] – apoCaM+myosin-5A 
[[2k0f]] – hCaM+myosin light chain kinase peptide – NMR 
[[2bbm]], [[2bbn]] - DmCaM+myosin light chain kinase 
[[2bki]], [[2bkh]] – CaM+myosin VI – pig 
[[1zuz]] – hCaM+DRP kinase peptide 
[[2bcx]] – cCaM+ryanodine receptor 1 peptide 
[[2fot]] - bCaM+α-II spectrin CBD 
[[2f2o]], [[2f2p]] – bCaM+calcineurin CBD 
[[2col]], [[2yrt]], [[2yru]] – BpCaM+adenyl cyclase – ''Bordetella pertussis'' 
[[1zot]] – BpCaM C-terminal+adenyl cyclase 
[[1xfu]], [[1xfv]], [[1xfw]], [[1xfx]], [[1xfy]], [[1xfz]], [[1y0v]], [[1sk6]], [[1pk0]], [[1lvc]], [[1k90]], [[1k93]] - CaM+adenyl cyclase – ''Bacillus anthracis'' 
[[1sy9]] – XlCaM+olfactory channel peptide 
[[1xa5]] – bCaM+KAR-2 
[[1l7z]] – hCaM+CAP-23/NAP-22 CBD 
[[1qs7]], [[1qtx]] – CaM+RS20 – ''Escherichia coli'' 
[[1vrk]] – CaM (mutant)+RS20 
[[1nwd]] – XlCaM+glutamate decarboxylase CBD 
[[1mxe]] – DmCaM+rCaMKI CBD 
[[1iq5]] – XlCaM+CaM dependent kinase CBD 
[[1cm1]], [[1cm4]], [[1cdm]] - bCaM+CaM dependent kinase CBD 
[[1cdl]] - hCaM+CaM dependent kinase CBD 
[[1ckk]] - XlCaM+CaM dependent kinase CBD - NMR 

==See Also==

* [http://en.wikipedia.org/wiki/Calmodulin Calmodulin at Wikipedia]
* [http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb44_1.html Molecule of the Month (08/2003) at RCSB PDB]
*{{Proteopedia|2fot}} page for Calmodulin crystal complex between calmodulin and alpha11-[[spectrin]]

== Bibliography ==
<references/>

Calmodulin

2010-10-26T09:39:11Z

Enrico Ravera:

Maximum Occurrence

2010-10-26T09:38:20Z

Enrico Ravera:

Maximum Occurrence (MO)<ref name='jacs_rav'>doi:10.1021/ja1063923</ref> refers to a method for making rigorous numerical assessments about the maximum percent of time that a conformer of a flexible macromolecule can exist and still be compatible with the experimental data. Maximum Occurrence of a conformer is defined as the maximum weight that it can have in one ensemble that matches the average experimental data.
== Background ==
Flexible proteins, even in the simplest case of two rigid domains linked by a flexible region, may sample a wide conformational space. Such variety makes their study by X-ray crystallography difficult, because a single conformation is trapped in the crystal, if crystals are obtained at all. Solution techniques such as Nuclear Magnetic Resonance (NMR) and Small-Angle Scattering of both X-rays and Neutrons (SAXS and SANS), provide experimental observables averaged over many conformation with different weights.
The problem of recovering from the data themselves the conformational ensemble that generated the data is an ill-defined inverse problem, that admits an infinite number of solutions. 
Popular methods for the determination of conformational disorder rely on the construction of ensembles that are solutions to this problem. Anyway, there is no proof that one solution can be better than another, so quantitative assessments are risky with these approaches. 
The Maximum Occurrence is the maximum percent of time that a conformer of a macromolecule can exist and still be compatible with the experimental data (see Figure 1). In this case, the quantitative assessment is rigorous, since only one conformer of the ensemble is considered while the completing conformers are not artificially endowed of physical relevance that they may not have.
[[Image:MO_profiles.png|thumbnail|350px|Figure 1: Maximum Occurrence profiles for some conformers of Calcium-loaded calmodulin. Dashed line indicates the maximum allowed disagreement between experimental and calculated data.]]
Recently, an implementation of this method based on distributed computing was presented to evaluate the MO profiles for a large number of conformers<ref name='jacs_rav'/>: this represents an evolution of a previous approach where few conformations with maximum allowed probability (MAP) were looked for<ref>DOI:10.1021/ja0726613</ref>.

== Methods for determination of Maximum Occurrence==

As already mentioned, to evaluate the Maximum Occurrence, an ensemble is sought so that the conformation under investigation is contained up to a certain value. At the Maximum Occurrence, no solution will be found fulfilling both the requisites of containing the desired conformation and of being compatible with the experimental data. Such process is exemplified in figure 2: the four boxes represent 4 different ensembles, containing the desired conformation (represented as a red star) at different weight (represented by the dimension of the red star): the fourth one is no more compatible with the experimental data.
[[Image:MO_ex_proteopedia.png|thumbnail|left|400px|Figure 2: Exemplification of the process of determination of maximum occurrence for a selected conformation.]]
The Maximum Occurrence approach has been developed using mainly paramagnetism-based NMR restraints and SAXS data, but it is well compatible with all the biophysical tools providing experimental data that are averages over all the states sampled by the system.
== Case Study: [[Calmodulin]] ==
Calmodulin (in this case N60D Calmodulin<ref>PMID:12834353</ref>(PDB ENTRIES [[1sw8]],[[2k0j]],[[2k61]])) is a two-domain protein experiencing high mobility in the central region<ref>PMID:1606151</ref><ref>PMID:3365370</ref>. Paramagnetic NMR restraints as pseudocontact shifts (PCS) and self-orientation residual dipolar couplings (RDC) provided further insight in the description of such conformational heterogeneity<ref>doi:10.1073/pnas.0308641101</ref>.
[[Image:Movie_MOforproteopedia.gif|thumbnail|400px|Figure 3: Orientation tensor representation for 400 conformational states of Calmodulin, color coded according to their MO values (from less than 5% in blue to more than 30% in red).To better explain their meaning, 10 randomly chosen models are shown as cartoons and then replaced by the three axes of their color-coded orientation tensors.]]
For this study, 400 conformers were chosen randomly, with the only requisite to be sterically allowed, and their maximum occurrence was evaluated against three sets of PCS (due to Tb3+,Dy3+ and Tm3+) and three sets of RDC (from the same metals), both measured at [http://www.cerm.unifi.it CERM (in Florence)], and X-rays scattering data up to 2nm-1, as recoded on the X33 beamline at [http://www.embl-hamburg.de/ExternalInfo/Research/Sax/ EMBL, DESY, Hamburg].
The results of the calculations are represented in figure 3: Orientation tensors, centered in the center of mass of the C-terminal domain, are shown color coded according to their MO value (from less than 5% in blue to more than 30% in red).

== Bibliography ==
<references/>

Maximum Occurrence

2010-10-26T09:21:41Z

Enrico Ravera: New page: Maximum Occurrence (MO)<ref>doi:10.1021/ja1063923</ref> refers to a method for making rigorous numerical assessments about the maximum percent of time that a conformer of a flexible macrom...

Maximum Occurrence (MO)<ref>doi:10.1021/ja1063923</ref> refers to a method for making rigorous numerical assessments about the maximum percent of time that a conformer of a flexible macromolecule can exist and still be compatible with the experimental data. Maximum Occurrence of a conformer is defined as the maximum weight that it can have in one ensemble that matches the average experimental data.
== Background ==
Flexible proteins, even in the simplest case of two rigid domains linked by a flexible region, may sample a wide conformational space. Such variety makes their study by X-ray crystallography difficult, because a single conformation is trapped in the crystal, if crystals are obtained at all. Solution techniques such as Nuclear Magnetic Resonance (NMR) and Small-Angle Scattering of both X-rays and Neutrons (SAXS and SANS), provide experimental observables averaged over many conformation with different weights.
The problem of recovering from the data themselves the conformational ensemble that generated the data is an ill-defined inverse problem, that admits an infinite number of solutions. 
Popular methods for the determination of conformational disorder rely on the construction of ensembles that are solutions to this problem. Anyway, there is no proof that one solution can be better than another, so quantitative assessments are risky with these approaches. 
The Maximum Occurrence is the maximum percent of time that a conformer of a macromolecule can exist and still be compatible with the experimental data (see Figure 1). In this case, the quantitative assessment is rigorous, since only one conformer of the ensemble is considered while the completing conformers are not artificially endowed of physical relevance that they may not have.
[[Image:MO_profiles.png|thumbnail|350px|Figure 1: Maximum Occurrence profiles for some conformers of Calcium-loaded calmodulin. Dashed line indicates the maximum allowed disagreement between experimental and calculated data.]]
Recently, an implementation of this method based on distributed computing was presented to evaluate the MO profiles for a large number of conformers: this represents an evolution of a previous approach where few conformations with maximum allowed probability (MAP) were looked for<ref>DOI:10.1021/ja0726613</ref>.

== Methods for determination of Maximum Occurrence==

As already mentioned, to evaluate the Maximum Occurrence, an ensemble is sought so that the conformation under investigation is contained up to a certain value. At the Maximum Occurrence, no solution will be found fulfilling both the requisites of containing the desired conformation and of being compatible with the experimental data. Such process is exemplified in figure 2: the four boxes represent 4 different ensembles, containing the desired conformation (represented as a red star) at different weight (represented by the dimension of the red star): the fourth one is no more compatible with the experimental data.
[[Image:MO_ex_proteopedia.png|thumbnail|left|400px|Figure 2: Exemplification of the process of determination of maximum occurrence for a selected conformation.]]
The Maximum Occurrence approach has been developed using mainly paramagnetism-based NMR restraints and SAXS data, but it is well compatible with all the biophysical tools providing experimental data that are averages over all the states sampled by the system.
== Case Study: [[Calmodulin]] ==
Calmodulin (in this case N60D Calmodulin<ref>PMID:12834353</ref>(PDB ENTRIES [[1sw8]],[[2k0j]],[[2k61]])) is a two-domain protein experiencing high mobility in the central region<ref>PMID:1606151</ref><ref>PMID:3365370</ref>. Paramagnetic NMR restraints as pseudocontact shifts (PCS) and self-orientation residual dipolar couplings (RDC) provided further insight in the description of such conformational heterogeneity<ref>doi:10.1073/pnas.0308641101</ref>.
[[Image:Movie_MOforproteopedia.gif|thumbnail|400px|Figure 3: Orientation tensor representation for 400 conformational states of Calmodulin, color coded according to their MO values (from less than 5% in blue to more than 30% in red).To better explain their meaning, 10 randomly chosen models are shown as cartoons and then replaced by the three axes of their color-coded orientation tensors.]]
For this study, 400 conformers were chosen randomly, with the only requisite to be sterically allowed, and their maximum occurrence was evaluated against three sets of PCS (due to Tb3+,Dy3+ and Tm3+) and three sets of RDC (from the same metals), both measured at [http://www.cerm.unifi.it CERM (in Florence)], and X-rays scattering data up to 2nm-1, as recoded on the X33 beamline at [http://www.embl-hamburg.de/ExternalInfo/Research/Sax/ EMBL, DESY, Hamburg].
The results of the calculations are represented in figure 3: Orientation tensors, centered in the center of mass of the C-terminal domain, are shown color coded according to their MO value (from less than 5% in blue to more than 30% in red).

== Bibliography ==
<references/>

2010-09-27T19:20:10Z

Enrico Ravera:

2010-09-22T14:37:28Z

Enrico Ravera:

Enrico Ravera, (Arezzo, Italy, 1st of April 1986)

Ph.D. student in Chemistry (Inorganic) at CERM and Department of Chemistry "Ugo Schiff", University of Florence.

Degrees:
2008. B.Sc. in Chemistry
2009. M.Sc. in Chemistry of Biological Molecules
*[[User:Enrico Ravera/Sandbox 1]]