Sandbox Reserved 199: Difference between revisions
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===History of NMR Ribonuclease Studies=== | ===History of NMR Ribonuclease Studies=== | ||
In 1957, Martin Saunder et al examined the structure of bovine pancreatic Ribonuclease using 1-Dimensional 1H NMR.<ref>Saunders, Martin. "The Nuclear | In 1957, Martin Saunder et al examined the structure of bovine pancreatic Ribonuclease using 1-Dimensional 1H NMR.<ref>Saunders, Martin, Arnold Wishnia, and John G. Kirkwood. "The Nuclear Magnetic Resonance Spectrum of Ribonuclease." Communications to the Editor 79; 20 May (1957). Print.</ref> | ||
In 1988, Udgaonkar et al. used 2-dimensional 1H NMR to study bovine pancreatic Ribonuclease and measured protein folding dynamics, which supported the framework model protein folding mechanism.<ref name="Udgaonkar"/> | In 1988, Udgaonkar et al. used 2-dimensional 1H NMR to study bovine pancreatic Ribonuclease and measured protein folding dynamics, which supported the framework model protein folding mechanism.<ref name="Udgaonkar"/> | ||
In 1993, Santoro et al. used 3-dimensional 1H NMR to study bovine pancreatic Ribonuclease to compare NMR structures with X-Ray Crystallography structures of RNase A.<ref name="Santoro"/> | In 1993, Santoro et al. used 3-dimensional 1H NMR to study bovine pancreatic Ribonuclease to compare NMR structures with X-Ray Crystallography structures of RNase A.<ref name="Santoro" /> | ||
In 2008, Rico et al. used 3-dimensional 1H NMR to study human pancreatic Ribonuclease. They looked at active site conformational changes upon substrate binding, and suggested possible biological implications.<ref name="rico"/> | In 2008, Rico et al. used 3-dimensional 1H NMR to study human pancreatic Ribonuclease. They looked at active site conformational changes upon substrate binding, and suggested possible biological implications.<ref name="rico"/> | ||
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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. | 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 wild-type | Starting with denatured wild-type RNaseA, 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> 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/ | <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_63/2'>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>, <scene name='Sandbox_Reserved_199/2aas_-_ile106/1'>Ile 106</scene>, and <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, β sheets were proposed to be 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' /> | ||
==Ribonuclease NMR Structure Versus X-Ray Crystallography Ribonuclease Structure<ref name"Santoro">PMID: 8381876 </ref>== | ==Ribonuclease NMR Structure Versus X-Ray Crystallography Ribonuclease Structure<ref name="Santoro">PMID: 8381876 </ref>== | ||
===Experimental Procedure=== | ===Experimental Procedure=== | ||
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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, 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/ | 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_47/2'>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_-_51-54/2'>Val 54 (NH)-Leu 51 (CO)</scene>, and <scene name='Sandbox_Reserved_199/2aas_-_73_63/2'>Cys 72 (NH)-Val 63 (CO)</scene>. The researchers suggested these differences are most likely due to the same pH phenomenon mentioned above. | ||
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/ | 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/3'>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 name="rico">PMID: 18495155</ref>== | ==Solution Structure and Dynamics of Human Pancreatic Ribonuclease<ref name="rico">PMID: 18495155</ref>== | ||
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===Experimental Procedure=== | ===Experimental Procedure=== | ||
Using 3D NMR, Rico et al. obtained the first NMR 3-dimensional structures of Human RNase 1. Using 20 different RNase 1 NMR structures, researchers compared the RMSD values of key RNase 1 residues. A residue with a high RMSD value has more flexibility and a residue’s ability to adapt to numerous conformations may be crucial to its active role within the enzyme. | |||
Furthermore, this study characterized the | Furthermore, this study characterized the dimerization of wt Ribonuclease and mutated Ribonuclease variants. Similar dimerization, observed in certain Bovine RNases, greatly enhances enzymatic activity and anti-tumoral action. Using varying enzyme concentrations, pH conditions and site-directed mutations, RNase 1 was dimerized. | ||
This study also compares the location of certain residues in | This study also compares the location of certain residues in bound RNase 1 to unbound RNase. This comparison, provides greater insight into the catalytic mechanism and substrate specificity of RNase 1. | ||
[[Image:Kroupa RNase Dimer2.png|thumb |left |alt=X-Ray Diffraction RNase Dimer. |3F8G-Domain swapped dimer of human pancreatic Ribonuclease I. Structure determined by X-Ray crystallography]] | [[Image:Kroupa RNase Dimer2.png|thumb |left |alt=X-Ray Diffraction RNase Dimer. |3F8G-Domain swapped dimer of human pancreatic Ribonuclease I. Structure determined by X-Ray crystallography]] | ||
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===Data and Results=== | ===Data and Results=== | ||
A 3D NMR structure was obtained with a backbone RMSD of 1.07Å. The obtained model shows a similar tertiary structure to the kidney bean shaped RNase A and is stabilized by four <scene name='Sandbox_Reserved_199/2k11_disulfide_bonds/1'>disulfide bonds</scene>. The structure shows three <scene name='Sandbox_Reserved_199/2k11_alpha_helix/2'>α-helices</scene> and seven <scene name='Sandbox_Reserved_199/2k11_beta_sheets/1'>β-sheets</scene>. While this structure matches up fairly well with previous X-Ray crystallography structures of RNase 1, important differences in residue positioning can be seen in the <scene name='Sandbox_Reserved_199/2k11_all_models/3'>3D NMR structure</scene> which are not apparent in X-Ray crystallography. Specifically, certain residues with more flexibility undergo a significant conformational change when bound to certain substrates, such as the human ribonuclease inhibitor ( | A 3D NMR structure was obtained with a backbone RMSD of 1.07Å. The obtained model shows a similar tertiary structure to the kidney bean shaped RNase A and is stabilized by four <scene name='Sandbox_Reserved_199/2k11_disulfide_bonds/1'>disulfide bonds</scene>. The structure shows three <scene name='Sandbox_Reserved_199/2k11_alpha_helix/2'>α-helices</scene> and seven <scene name='Sandbox_Reserved_199/2k11_beta_sheets/1'>β-sheets</scene>. While this structure matches up fairly well with previous X-Ray crystallography structures of RNase 1, important differences in residue positioning can be seen in the <scene name='Sandbox_Reserved_199/2k11_all_models/3'>3D NMR structure</scene> which are not apparent in X-Ray crystallography. Specifically, certain residues with more flexibility undergo a significant conformational change when bound to certain substrates, such as the human ribonuclease inhibitor (hRI). These residues include: <scene name='Sandbox_Reserved_199/2k11_flexible_residues/1'>Arg 4, Lys 6, Arg 32, Arg 39, and Lys 102</scene>. | ||
This | This large conformational change suggests an “induced-fit” model of substrate binding and may prove vital to fully understanding RNase 1’s binding specificity for Hcrl; although two residues, <scene name='Sandbox_Reserved_199/2k11_42_43/1'>Pro 42 and Val43</scene>, show much more rigidity and possibly contribute some “lock-and-key” binding interaction. Designing cytotoxic variants of RNase1 may prove difficult due to the large number of active amino acids; numerous mutations may be required to fully "kill" the enyzme. | ||
[[Image:Kroupa Ribonuclease inhibitor.png|thumb |right |alt=RNase Inhibitor. |Example Ribonuclease inhibitor structure<ref>Willow. Top view of ribbon diagram of ribonuclease inhibitor (PDB accession code 2BNH). Made with MOLMOL. 2006. Web. 1 Apr. 2011..</ref>.]] | [[Image:Kroupa Ribonuclease inhibitor.png|thumb |right |alt=RNase Inhibitor. |Example Ribonuclease inhibitor structure<ref>Willow. Top view of ribbon diagram of ribonuclease inhibitor (PDB accession code 2BNH). Made with MOLMOL. 2006. Web. 1 Apr. 2011..</ref>.]] | ||
15N NMR relaxation shows increased T1 values for the residues found in these sheets and loops (0.63-0.64s relative to 0.60s in helices) | 15N NMR relaxation shows increased T1 values for the residues found in these sheets and loops (0.63-0.64s relative to 0.60s in helices), suggesting greater flexibility in these regions as well. | ||
Interestingly, the global correlation time of RNase 1 was | Interestingly, the global correlation time of RNase 1 was much longer than expected for a 13.7 kDa monomer (10ns compared to 6-7ns) indicating the RNase1 undergoes dimerization forming a monomer/dimer equilibrium. Because dimerization affects the enzymatic activity of bovine ribonuclease, RNase 1 variants with increased dimerization may be potential anti-cancer therapeutics. | ||
== References == | == References == | ||
<references /> | <references /> | ||