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===Overview===
===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 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. 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.


3D 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).
3D 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===
===NMR Versus X-Ray Crystallography===
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[[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.]]


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.
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.
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