Sandbox Reserved 199: Difference between revisions

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===Medical Significance===
===Medical Significance===


Ribonucleases show specific toxicity to tumor cells. In fact, Onconase ® (an RNase A homolog from the [http://en.wikipedia.org/wiki/Northern_Leopard_Frog Northern Leopard Frog]) is already in clinical trials due to its toxicity to cancer cells. However, due to possible immunogenicity of the frog enzyme, much effort has been focused on developing a mutated human RNase which can prevent inhibition by human ribonuclease inhibitor (HcRI).  HcRI selectively binds to human pancreatic RNase (RNase 1) and impedes its enzymatic activity.  Without sufficient degradation of mRNA, the cell undergoes enhanced gene expression and can lead to cancer.  Correctly characterizing RNase 1’s structure and binding specificity via NMR will prove vital to the development of RNase-based anti-cancer treatments.  
Ribonucleases show specific toxicity to tumor cells. In fact, Onconase ® (an RNase A homolog from the [http://en.wikipedia.org/wiki/Northern_Leopard_Frog Northern Leopard Frog]) is currently in phase IIIb clinical trials for the treatment of malignant mesothelioma. However, due to possible immunogenicity of the frog enzyme, much effort has been focused on developing a cytotoxic human RNase, which evades inhibition by human ribonuclease inhibitor (HRI).  HRI selectively binds to human pancreatic RNase (RNase 1) and impedes its enzymatic activity.  Correctly characterizing the structure and binding specificity of RNase via NMR could assist in the development of RNase-based anti-cancer treatments.  


==NMR Study of Ribonuclease Folding Dynamics<ref> Udgaonkar, Jayant B., and Robert L. Baldwin. "NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A." Nature 335.1; 20 Oct. (1988). Print.  </ref>==
==NMR Study of Ribonuclease Folding Dynamics<ref> Udgaonkar, Jayant B., and Robert L. Baldwin. "NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A." Nature 335.1; 20 Oct. (1988). Print.  </ref>==
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===Experimental Procedure===
===Experimental Procedure===


Using 2-dimensional 1H NMR, Udgaonkar et al. studied the folding pathway of bovine pancreatic Ribonuclease using an [http://en.wikipedia.org/wiki/Hydrogen-deuterium_exchange exchange reaction] between <scene name='Sandbox_Reserved_199/2aas_-_backbone_nitrogens/2'>deuterated peptide backbone amide protons</scene>  with solvent protons.  2- dimensional 1H NMR allowed for monitoring of proton exchange in the amide backbone for ten second time intervals, and this proton labeling could be terminated via a rapid drop in pH reaction conditions.  This research focused on initial protein folding steps.
Using 2-dimensional 1H NMR, Udgaonkar et al. studied the folding pathway of bovine pancreatic Ribonuclease using an [http://en.wikipedia.org/wiki/Hydrogen-deuterium_exchange exchange reaction] between <scene name='Sandbox_Reserved_199/2aas_-_backbone_nitrogens/2'>deuterated peptide backbone amide protons</scene>  with solvent protons.  2- dimensional 1H NMR allowed for monitoring of proton exchange in the amide backbone for ten second time intervals, and this proton labeling could be terminated via a rapid drop in pH reaction conditions.  This research focused on the initial protein folding steps.


Starting with denatured wt Ribonuclease, it was suggested that as the peptide began to fold, the backbone amide proteins would become less energetically favorable to exchange protons with the solvent as the backbone amide protons became involved in folding-related intermolecular interactions (such as <scene name='Sandbox_Reserved_199/2aas_-_backbone_hydrogen_bondi/1'>hydrogen bonding</scene> ).
Starting with denatured wild-type RNase, it was hypothesized that as the peptide began to fold, the backbone amide proteins would become less energetically favorable to exchange protons with the solvent as the backbone amide protons became involved in folding-related intermolecular interactions (such as <scene name='Sandbox_Reserved_199/2aas_-_backbone_hydrogen_bondi/1'>hydrogen bonding</scene> ).


===Data and Results===
===Data and Results===


<scene name='Sandbox_Reserved_199/2aas_-_five_amide_backbone_pro/3'>Five backbone amide protons</scene> became evident as those involved in folding-related intermolecular interactions during initial protein folding steps: Those associated with <scene name='Sandbox_Reserved_199/2aas_-_val_63/1'>Val 63</scene>, <scene name='Sandbox_Reserved_199/2aas_-_val_118/1'>Val 118</scene>, <scene name='Sandbox_Reserved_199/2aas_-_ile81/2'>Ile 81</scene>, <scene name='Sandbox_Reserved_199/2aas_-_thr82/1'>Thr 82</scene>, and <scene name='Sandbox_Reserved_199/2aas_-_ile106/1'>Ile 106</scene>.  All five of these protons are involved in hydrogen bonding within the <scene name='Sandbox_Reserved_199/2aas_-_beta_sheet/2'>β sheet secondary structure</scene> of Ribonuclease; therefore, it was believed that this secondary structure was the starting point for the folding mechanism of Ribonuclease.  Furthermore, this suggests the formation of a stable secondary structure before the formation of the <scene name='Sandbox_Reserved_199/2aas_-_final_tertiary_structue/1'>final tertiary structure</scene>, which is consistent with the framework model of [http://en.wikipedia.org/wiki/Protein_folding#Protein_nuclear_magnetic_resonance_spectroscopy protein folding mechanisms] (in comparison with the jigsaw puzzle model).
<scene name='Sandbox_Reserved_199/2aas_-_five_amide_backbone_pro/3'>Five backbone amide protons</scene> were shown to be involved in folding-related intermolecular interactions during initial protein folding steps: amide protons from <scene name='Sandbox_Reserved_199/2aas_-_val_63/1'>Val 63</scene>, <scene name='Sandbox_Reserved_199/2aas_-_ile81/2'>Ile 81</scene>, <scene name='Sandbox_Reserved_199/2aas_-_thr82/1'>Thr 82</scene>, and <scene name='Sandbox_Reserved_199/2aas_-_ile106/1'>Ile 106</scene>, <scene name='Sandbox_Reserved_199/2aas_-_val_118/1'>Val 118</scene>.  All five of these protons are involved in hydrogen bonding within the <scene name='Sandbox_Reserved_199/2aas_-_beta_sheet/2'>β sheet secondary structure</scene> of Ribonuclease; therefore, it was believed that this secondary structure was the starting point for the folding mechanism of Ribonuclease.  Furthermore, this supports the formation of a stable secondary structure before the formation of the <scene name='Sandbox_Reserved_199/2aas_-_final_tertiary_structue/1'>final tertiary structure</scene>, which is consistent with the framework model of [http://en.wikipedia.org/wiki/Protein_folding#Protein_nuclear_magnetic_resonance_spectroscopy protein folding mechanisms].


<Structure load='2AAS' size='350' frame='true' align='right' caption='2AAS - NMR Scructure of Bovine Pancreatic Ribonuclease' scene='Sandbox_Reserved_199/2aas_-_all_models/4' />
<Structure load='2AAS' size='350' frame='true' align='right' caption='2AAS - NMR Scructure of Bovine Pancreatic Ribonuclease' scene='Sandbox_Reserved_199/2aas_-_all_models/4' />
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The NMR experiment yielded an overall R-Factor of 0.44, compared to an X-Ray Crystallographic R-Factor of 0.45.  This means that the NMR structure shows a higher structural reliability compared to the X-Ray Crystallographic structure.
The NMR experiment yielded an overall R-Factor of 0.44, compared to an X-Ray Crystallographic R-Factor of 0.45.  This means that the NMR structure shows a higher structural reliability compared to the X-Ray Crystallographic structure.
Overall, the bovine Ribonuclease NMR tertiary structure matches closely the corresponding X-Ray Crystallography structure.  The overall shape, main-chain fold, and side chain positions of most residues are similar between the structures of the two methods.  Experimentally determined tertiary structural differences between the two methods were suggested to be due to pH differences, crystal packing, solvation, and temperature variability.
Overall, the NMR tertiary structure of RNase matches closely with the corresponding X-Ray crystallography structure.  The overall shape, main-chain fold, and side chain positions of most residues are similar between the two structures.  Experimentally determined tertiary structural differences between the two methods were suggested to be due to pH differences, crystal packing, solvation, and temperature variability.


Previously, researchers found the side chain position of <scene name='Sandbox_Reserved_199/2aas_-_his_119/1'>His 119</scene> in the enzyme’s <scene name='Sandbox_Reserved_199/2aas_-_all_models/5'>active site</scene> (<scene name='Sandbox_Reserved_199/2aas_-_active_site_space_fill/1'>spacefill</scene>) of NMR structures to be different than that of X-Ray Crystallography studies.  Crystals show a static position of this His 119 residue, yet NMR structures suggest a dynamic equilibrium between the two conformational puckers of the <scene name='Sandbox_Reserved_199/2aas_-_his_119_imidazole/1'>His 119 imidazole ring</scene>.  This single residue difference between crystal and solution studies amplifies to cause a major difference in surrounding amino acid residues: <scene name='Sandbox_Reserved_199/2aas_-_residue_4/1'>4</scene>, <scene name='Sandbox_Reserved_199/2aas_-_residue_4_106_107_108/1'>106-108</scene>, and <scene name='Sandbox_Reserved_199/2aas_-_residue_4_106_107_108_1/1'>116-118</scene>.  The researchers proposed that this difference is most likely due to pH induced charge repulsion of His 119 with <scene name='Sandbox_Reserved_199/2aas_-_asp_14/1'>Asp 14</scene> and <scene name='Sandbox_Reserved_199/2aas_-_his_48/2'>His 48</scene> in solution.
Previously, researchers found the side chain position of <scene name='Sandbox_Reserved_199/2aas_-_his_119/1'>His 119</scene> in the enzyme’s <scene name='Sandbox_Reserved_199/2aas_-_all_models/5'>active site</scene> (<scene name='Sandbox_Reserved_199/2aas_-_active_site_space_fill/1'>spacefill</scene>) of NMR structures to be different than that of X-Ray Crystallography studies.  Crystals show a static position of this His 119 residue, yet NMR structures suggest a dynamic equilibrium between the two conformational puckers of the <scene name='Sandbox_Reserved_199/2aas_-_his_119_imidazole/1'>His 119 imidazole ring</scene>.  This single residue difference between crystal and solution studies amplifies to cause a major difference in surrounding amino acid residues: <scene name='Sandbox_Reserved_199/2aas_-_residue_4/1'>4</scene>, <scene name='Sandbox_Reserved_199/2aas_-_residue_4_106_107_108/1'>106-108</scene>, and <scene name='Sandbox_Reserved_199/2aas_-_residue_4_106_107_108_1/1'>116-118</scene>.  The researchers proposed that this difference is most likely due to pH induced charge repulsion of His 119 with <scene name='Sandbox_Reserved_199/2aas_-_asp_14/1'>Asp 14</scene> and <scene name='Sandbox_Reserved_199/2aas_-_his_48/2'>His 48</scene> in solution.


More than 60 main-chain hydrogen bonds were observed, which closely corresponds to the number of hydrogen bonds determined in crystals; however, there exist a few discrepancies.  In the NMR structure, there was determined to be a hydrogen bond between the amide proton on <scene name='Sandbox_Reserved_199/2aas_-_17_14_dbl/1'>Thr 17 (NH)-Asp 14 (CO) carbonyl</scene>, as well as between <scene name='Sandbox_Reserved_199/2aas_-_49_47/1'>Glu 49 (NH)-Val 47 (CO)</scene>.  The researchers suggested these differences are most likely due to the same pH phenomenon mentioned above.  Other hydrogen bonds present in the NMR structure but not present in the crystal structure are: <scene name='Sandbox_Reserved_199/2aas_-_32_28/1'>Ser 32 (NH)-Gln 28 (CO)</scene>, <scene name='Sandbox_Reserved_199/2aas_-_51-54/1'>Val 54 (NH)-Leu 51 (CO)</scene>, and <scene name='Sandbox_Reserved_199/2aas_-_73_63/1'>Cys 72 (NH)-Val 63 (CO)</scene>.
More than 60 main-chain hydrogen bonds were observed, which closely corresponds to the number of hydrogen bonds determined in crystals; however, a few discrepancies existed such as hydrogen bonds between the amide proton on <scene name='Sandbox_Reserved_199/2aas_-_17_14_dbl/1'>Thr 17 (NH)-Asp 14 (CO) carbonyl</scene>, <scene name='Sandbox_Reserved_199/2aas_-_49_47/1'>Glu 49 (NH)-Val 47 (CO)</scene>, <scene name='Sandbox_Reserved_199/2aas_-_32_28/1'>Ser 32 (NH)-Gln 28 (CO)</scene>, <scene name='Sandbox_Reserved_199/2aas_-_51-54/1'>Val 54 (NH)-Leu 51 (CO)</scene>, and <scene name='Sandbox_Reserved_199/2aas_-_73_63/1'>Cys 72 (NH)-Val 63 (CO)</scene>.  The researchers suggested these differences are most likely due to the same pH phenomenon mentioned above.


The researchers also utilized the NMRs advantage of determining flexibility of Ribonuclease.  Overall, the largest conformational flexibility resulted within the side-chains.  Specifically, side-chain mobility is greatest in residues  <scene name='Sandbox_Reserved_199/2aas_-_side_chain_flexibility/1'>1, 7, 15, 18, 24, 37, 59, 66, 94, 123, and 124</scene> (shown in white).  As expected, the backbone torsion angles were seen to be more rigid (less conformational flexibility) within the <scene name='Sandbox_Reserved_199/2aas_-_active_site_rigidity/1'>active site (shown in white)</scene> of Ribonuclease.
The NMR structure also highlighted flexibility of RNase A.  Overall, the largest conformational flexibility was found in the side-chains.  Specifically, side-chain mobility is greatest in residues  <scene name='Sandbox_Reserved_199/2aas_-_side_chain_flexibility/1'>1, 7, 15, 18, 24, 37, 59, 66, 94, 123, and 124</scene> (shown in white).  As expected, the backbone torsion angles were seen to be more rigid (less conformational flexibility) within the <scene name='Sandbox_Reserved_199/2aas_-_active_site_rigidity/1'>active site (shown in white)</scene> of RNase A.


==Solution Structure and Dynamics of Human Pancreatic Ribonuclease<ref> Rico, M. "The Solution Structure and Dynamics of Human Pancreatic Ribonuclease Determined by NMR Spectroscopy Provide Insight into Its Remarkable Biological Activities and Inhibition." Journal of Molecular Biology 379; 14 Apr. (2008). Print. </ref>==
==Solution Structure and Dynamics of Human Pancreatic Ribonuclease<ref> Rico, M. "The Solution Structure and Dynamics of Human Pancreatic Ribonuclease Determined by NMR Spectroscopy Provide Insight into Its Remarkable Biological Activities and Inhibition." Journal of Molecular Biology 379; 14 Apr. (2008). Print. </ref>==
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