Sandbox Reserved 199: Difference between revisions

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OCA, Daniel Kroupa

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-[[Image:Kroupa RNase.png|border |400px  |right |alt=NMR. |2K11-NMR structure of bovine pancreatic RNase.]]
+[[Image:Kroupa RNase Rainbox.png|thumb |400px  |right |alt=NMR. |2AAS-NMR structure of bovine pancreatic ribonuclease A. Color coded by rainbow gradient typical to NMR sturctures with amino (blue) to carboxy (red).  Active site shown in white. ]]
 == Introduction ==
-[[Image:Kroupa RNase Cartoon 2.png|thumb |Right|alt=NMR Cartoon. |Cartoon NMR structure of bovine pancreatic Ribonuclease.]]
+[[Image:Kroupa RNase Cartoon 2.png|thumb |Right|alt=NMR Cartoon. |2AAS-Cartoon NMR structure of bovine pancreatic Ribonuclease A.]]
 ===Overview===
-Ribonucleases are some of the most well studied enzymes within the scientific community due to their ample availability as well as their significant role within a cell. In the past, 3D NMR structures of Bovine Pancreatic Ribonuclease (RNase A) and Human Pancreatic Ribonuclease (RNase 1) were obtained. While structures of RNase A and RNase 1 via X-Ray crystallography have been around for some time, the 3D NMR structures present much more information on specific locations of side chain residues as well as their flexibility in the unbound enzymes. Because NMR does not require a "frozen" crystal structure (X-Ray crystallography), NMR imaging can show much more accurate detail into the actual, solution enzyme (folding, flexibility etc.)
+Ribonuclease A has served as a model for protein structure and finction, and was the third enzyme whose three-dimensional structure was determined.<ref name="raines"> Raines, Ronald T. "Ribonuclease A." Chemical Reviews;  98, 1045-1065 (1998). Print.</ref>  Ribonuclease has been called the most studies enzyme of the 20th century due to its ample availability as well as its significant role within the cell.<ref name="raines"/>  While the structures of bovine pancreatic ribonuclease (RNase A) and human pancreatic ribonuclease (RNase 1) determined by X-Ray crystallography have been around for some time, the 3D NMR structures of present provide much more information on specific locations of side chain residues and their flexibility. Because NMR does not require a "frozen" crystal structure, NMR imaging can show much more accurate detail into the actual, solution enzyme (folding, flexibility etc.)
-Ribonuclease A and Ribonuclease 1 are both good targets for 3D NMR. Not only are they small proteins which make NMR a more feasible option, they also have numerous characteristics that can only be observed in an uncrystallized state, such as internal flexibility and 3D domain swapping.
+Ribonuclease A and Ribonuclease 1 are both good targets for 3D NMR. Not only are they small proteins (~13 KDa), they also have numerous characteristics that are observable only by NMR, such as internal flexibility and 3D domain swapping.
-D NMR spectroscopy has had shed light on protein folding dynamics as a whole, suggesting a framework model of folding (Folding order = primary structure, secondary structure, tertiary structure).
+D NMR spectroscopy has shed light on protein folding dynamics as a whole, suggesting a framework model of folding.  In other words, Ribonuclease NMR studies have provided evidence that secondary structure within the protein fold before the tertiary folds begin (Folding order = primary structure, secondary structure, tertiary structure).
 ===NMR Versus X-Ray Crystallography===
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 The two predominant methods of protein tertiary structure determination are [http://www.proteopedia.org/wiki/index.php/X-ray_crystallography X-Ray Crystallography]and [http://www.proteopedia.org/wiki/index.php/NMR Nuclear Magnetic Resonance](NMR) Spectroscopy.
-[[Image:Kroupa X-Ray.jpg|thumb |left |alt=X-Ray Diffraction image. |X-Ray Diffration pattern of a crystallized SARS protease at 2.1 Angstrom resolution.]]
+[[Image:Kroupa X-Ray.jpg|thumb |left |alt=X-Ray Diffraction image. |X-Ray Diffration pattern of a crystallized SARS protease at 2.1 Angstrom resolution.<ref>Dahl, Jeff. X-ray diffraction pattern of crystallized 3Clpro, a SARS protease. (2.1 Angstrom resolution). 2006. Web. 1 Apr. 2011.</ref>]]
-X-Ray Crystallography entails protein purification, crystallization of the protein, collection of X-Ray diffraction data, calculation of the protein’s electron density relative to the determined data, and finally fitting the protein’s determined residue sequence into the electron density.  Crystallization of the protein often lends itself to being the most challenging aspect of this method.  While any size protein can be studied via X-Ray Crystallography and the method is well-established, it is often difficult to perform for membrane proteins and the data received reveals no information about the protein’s hydrogen atoms.  Also, an assumption made for X-Ray Crystallographic studies is that the crystallized protein is in a conformation similar to that seen in solution.  For more information regarding X-Ray Crystallography please click [http://en.wikipedia.org/wiki/X-ray_crystallography here].
+To complete an X-Ray Crystallographic structure of a protein requires protein purification, crystallization of the protein, collection of X-Ray diffraction data, calculation of the protein’s electron density, and finally fitting the protein’s determined amino acid sequence into the electron density.  Crystallizing the protein is often the rate-limiting step in structure determination.  While any size protein can be studied via X-Ray Crystallography and the method is well-established, it is often difficult to crystallize membrane proteins, hydrogen atoms are not present, and crystal conditions are assumed to mimic solution conditions.  For more information regarding X-Ray Crystallography please click [http://en.wikipedia.org/wiki/X-ray_crystallography here].
-Bimolecular NMR Spectroscopy involves protein purification, dissolving the protein in a suitable solvent, collecting the NMR data, assigning NMR signals, and finally calculating the protein’s tertiary structure.  With NMR, the most difficult step is often correctly assigning NMR signals.  Although there is no need to crystallize the protein of interest and most hydrogen atoms are evident, NMR is difficult for proteins that don’t dissolve well in common solvents and works best for small proteins.  1-dimensional, 2-dimensional, and 3-dimensional NMR spectroscopy is readily available; however, 2-dimensional and 3-dimensional NMR is most often used for protein tertiary structure determination.  2D NMR reveals chemical shift correlations between spinnable nuclei such as 1H, 13C, 15N, and 13P, as well as atomic coupling, or proximity correlations via bonding.  3D NMR utilizes this methodology in addition to detection of another nuclear spin phenomenon known as the [http://en.wikipedia.org/wiki/Nuclear_Overhauser_effect Nuclear Overhauser Effect](NOE), in which proximity correlations are can be observed in 3D space.
+To complete a bi-molecular NMR Spectroscopy structure of a protein requires protein purification, dissolving the protein in a suitable solvent, collecting the NMR data, assigning NMR signals, and finally calculating the protein’s tertiary structure.  With NMR, the most difficult step is often correctly assigning NMR signals.  Although there is no need to crystallize the protein of interest and most hydrogen atoms are present, NMR is difficult for proteins that do not dissolve well in common solvents and works best for small proteins.  1-dimensional, 2-dimensional, and 3-dimensional NMR spectroscopy is readily available; however, 2-dimensional and 3-dimensional NMR are most often used for protein tertiary structure determination.  2D NMR reveals chemical shift correlations between spinnable nuclei such as 1H, 13C, 15N, and 13P, as well as atomic coupling, or proximity correlations via bonding.  3D NMR utilizes this methodology in addition to detection of another nuclear spin phenomenon known as the [http://en.wikipedia.org/wiki/Nuclear_Overhauser_effect Nuclear Overhauser Effect](NOE), in which proximity correlations are can be observed in 3D space.
-[[Image:Kroupa 2d NMR.png|thumb |right|alt=2D NMR Image. |2D COSY NMR spectrum of progesterone.]]
+[[Image:Kroupa 2d NMR.png|thumb |right|alt=2D NMR Image. |2D COSY NMR spectrum of progesterone<ref>English Wikipedia. COSY NMR spectrum progesterone. Web. 1 Apr. 2011.</ref>.]]
-Due to the complexity of assigning NMR signals to specific protons, the data gathered is usually put into a computer that uses complex computer algorithms to render a protein’s tertiary structure.  Even though NMR often requires more prior knowledge of the studied protein’s structural information, the end result is often more telling than that of X-Ray Crystallography.  With NMR, one can gain a better insight as to what exactly a folded protein might look like in solution including the protein’s conformational flexibility.  An example <scene name='Sandbox_Reserved_199/2aas_-_all_models/4'>NMR structure revealing the flexibility of a protein</scene> is the many-conformational structure of bovine pancreatic Ribonuclease seen when the page originally loads.  For more information regarding NMR Spectroscopy, click [http://en.wikipedia.org/wiki/Protein_nuclear_magnetic_resonance_spectroscopy here].
+Due to the complexity of assigning NMR signals to specific protons, complex computer algorithms are required to render a protein’s tertiary structure.  Even though NMR often requires more prior knowledge of the studied protein’s structural information, the end result contains more information about the solution structure of the protein including the protein’s conformational flexibility (see <scene name='Sandbox_Reserved_199/2aas_-_all_models/4'>bovine pancreatic Ribonuclease</scene>.  For more information regarding NMR Spectroscopy, click [http://en.wikipedia.org/wiki/Protein_nuclear_magnetic_resonance_spectroscopy here].
 ===History of NMR Ribonuclease Studies===
-In 1957, the first work was published examining the structure of bovine pancreatic Ribonuclease using 1-Dimensional 1H NMR by Martin Saunder et al.
+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 examined protein folding dynamics, which supported the framework model protein folding mechanism.
+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.
+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 of their findings.
+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"/>
 ===Medical Significance===
-Ribonucleases which can prevent inhibition by ribonuclease inhibitors have been looked at as potential anti-tumor agents for some time now. In fact, Onconase ® (an RNase A homolog from the green 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 name="Udgaonkar">PMID: 2845278 </ref>==
 ===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 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===
-<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_63/2'>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' />
-==Ribonuclease NMR Structure Versus X-Ray Crystallography Ribonuclease Structure<ref> Santoro, Jorge. "High-resolution Three-dimensional Structure of Ribonuclease A in Solution by Nuclear Magnetic Resonance Spectroscopy." Journal of Molecular Biology 229 (1993). Print.  </ref>==
+==Ribonuclease NMR Structure Versus X-Ray Crystallography Ribonuclease Structure<ref name="Santoro">PMID: 8381876 </ref>==
 ===Experimental Procedure===
@@ Line 71: / Line 71: @@
 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.
-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_47/2'>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 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/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> 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 name="rico">PMID: 18495155</ref>==
 <Structure load='2k11' size='350' frame='true' align='right' caption='2K11 - NMR Structure of Human Pancreatic Ribonuclese' scene='Sandbox_Reserved_199/2k11_all_models/2' />
@@ Line 85: / Line 85: @@
 ===Experimental Procedure===
-This study builds on the results of the previously mentioned experiment. Using 3D NMR, Rico et al. obtained the first NMR 3-dimensional structures of Human Rnase 1. Using 20 different RNase 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.
+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 dimerizational prperties of wt Ribonuclease and mutated Ribonuclease variants.  Similar dimerization, observed in certain Bovine RNases, has shown to greatly enhance enzymatic activity and have augmented anti-tumoral action. Using varying enzyme concentrations and pH conditions as well as site-directed mutations, researchers were able to promote dimerization of RNase 1 variants.
+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 HcrI bound RNase 1 to unbound RNase. Through this comparison, these researchers provide greater insight into the catalytic mechanism and substrate specificity of RNase 1.
+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 Dimer.png|thumb |left |alt=X-Ray Diffraction RNase Dimer. |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]]
 ===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 (HcrI).  These residues include: <scene name='Sandbox_Reserved_199/2k11_flexible_residues/1'>Arg 4, Lys 6, Arg 32, Arg 39, and Lys 102</scene>.
+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 data 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.
+	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.]]
+[[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>.]]
-N 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). This suggests greater flexibility in these regions as well.
+N 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 shown to be much longer than what is expected for a 13.7 kDa monomer (10ns compared to 6-7ns). This shows that under NMR sample conditions, the RNase1 undergoes dimerization forming a monomer/dimer equilibrium.  Because dimerization has shown to have a significant effect on enzyme activity in bovine RNases, certain mutated RNase 1’s which show increased dimerization may be potential anti-cancer therapeutics.
+	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 ==
-<ref group="xtra">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><references group="xtra"/>
 <references />