Sandbox Reserved 192: Difference between revisions

RNase A has been used as a foundation enzyme for study due to its stability, small size, and because its three-dimensional structure is fully determined by its amino acid sequence, needing no molecular chaperones. The 1972 Nobel Prize in Chemistry was awarded to three researchers for their work with RNase A on the folding of chains in RNase A and the stability of RNase A. The previously mentioned Christian Anfinsen received the 1972 Nobel Prize in Chemistry for his paper "Principles that govern the folding of protein chains." Stanford Moore and William H. Stein received the 1972 Nobel Prize in Chemistry for their paper "The chemical structures of pancreatic ribonuclease and deoxyribonuclease." The 1984 Nobel Prize in Chemistry was awarded to Robert Bruce Merrifield for his paper "Solid-phase synthesis" using RNase A (Raines). RNase A was the first enzyme and third protein for which its amino acid sequence was correctly determined and the third enzyme and fourth protein whose three-dimensional structure was determined by X-ray diffraction analysis [http://en.wikipedia.org/wiki/X-ray_diffraction_analysis]. Disulfide bonds in RNase A were determined after developing a method using Fast Atom Bombardment Mass Spectrometry (FABMS) [http://en.wikipedia.org/wiki/Fast_atom_bombardment]. The methods of NMR spectroscopy [http://en.wikipedia.org/wiki/NMR_spectroscopy] and Fourier transform infrared (FTIR) spectroscopy [http://en.wikipedia.org/wiki/Fourier_transform_infrared_spectroscopy]  were developed with RNase A in determining protein structure and protein folding pathways. These new methods, developed with RNase A, could be used for further research with other proteins (Raines).

Another member in the ribonuclease family and structural homologue to bovine RNase A is frog onconase [http://en.wikipedia.org/wiki/Onconase] or ONC. ONC is found in oocytes [http://en.wikipedia.org/wiki/Oocytes] and early embryos of northern leopard frogs. The frog ribonuclease variant shows both cytostatic (cell growth suppression) and cytotoxic (prevents cell divisions) characteristics when it interacts with tumor cells. According to Gahl et al. (2008), no side effects have been determined for ONC. Leland et al. (2001) looked to determine the interactions that control the folding of ONC in order to develop effective mimics of ONC. In order to determine the interactions that controlled folding, the regeneration of RNase A was studied. Although RNase A and ONC were structurally very similar, there were significant differences in their folding pathways. While ONC forms a stable disulfide intermediate, RNase A does not. ONC was also found to be missing a disulfide bond that RNase A possesses. In the case of both enzymes, entropy is lost in the formation of the disulfide bonds, but folding may be driven by enthalpically favorable interactions of the side chains. Further experiments are being done to identify intramolecular interactions that account for the increased rate and formation of the structured intermediate in ONC (Gahl).

The phenylalanine-46 (Phe46) residue located within the hydrophobic core of RNase A was experimentally replaced with other hydrophobic residues; leucine, valine and alanine. The goal was to conclude how the change would affect the conformational stability. It was concluded that the replacement of Phe46, which is key to the formation of the hydrophobic core, causes the destabilization of the RNase A by preventing the core from being tightly packed. The amino acids that are hyhdrophobic are; valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and cysteine. The protein folds with its hydrophobic amino acids facing inward and its hydrophilic amino acids facing outward to reduce the amount of water that interacts with the least number of hydrophobic residues (Kadonosono).