RNaseA Nobel Prizes: Difference between revisions

Ribonuclease A has been the subject of Nobel Prizes on Protein Folding and Solid Phase Peptide Synthesis.<ref name="Raines"> PMID:11848924</ref> The observation of ribonuclease folding helped Christian Anfinsen win the Nobel Prize in 1972 for his work on protein folding <ref>'Anfinsen Nobel Lecture' [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-lecture.html]</ref>. The presence of four disulfide bonds and two ''cis'' proline residues in the structure of RNase A greatly affects the structure and folding kinetics of RNase A <ref>'Anfinsen Nobel Biography' [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]</ref>. When RNase A undergoes reductive denaturation, it spontaneously folds back on itself to form the same structure.  The development of solid phase synthesis by Bruce Merrifield (Nobel Prize 1984) was a radical departure from traditional methods of bio-molecular synthesis that greatly increased efficiency. His method made possible the syntheses of much larger and more complex molecules; however, solid phase synthesis was not fully embraced until he demonstrated its full ability with the complete synthesis of Ribonuclease A.[http://nobelprize.org/nobel_prizes/chemistry/laureates/1984/merrifield-lecture.pdf]

Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding].  Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. Although RNase A has 105 possible disulfide bond pairings, only one set of four bonds occurs. This unique observation leads to the "thermodynamic hypothesis", that a protein's native state is determined by the thermodynamic favorability of the whole system; thus the tertiary structure must be predetermined by intramolecular interactions within the amino acid sequence.<ref>PMID: 4124164</ref> Since thermodynamic stability of a protein is affected by the environment's temperature, pH, and ionic strength, among other factors, the protein structure can only exist under physiological conditions. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding. Among the most important attributes of this model are noncovalent interactions, proline conformation, and disulfide bonding <ref name = 'Lehninger'>'Lehninger A., Nelson D.N, & Cox M.M. (2008) Lehninger Principles of Biochemistry. W. H. Freeman, fifth edition.' </ref>.

Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly.  This abnormality can be toxic to human nerve cells.  All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2' target='0'>hydrophobic and hydrophilic residues</scene>.  The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3' target='0'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque.  In the future researchers hope to design drugs that combat mistakes in protein folding <ref>'Hogan, Dan. ed. Dysfunctional Protein Dynamics Behind Neurological Disease?  ScienceDaily.2 Nov. 2009. www.sciencedaily.com'[http://www.sciencedaily.com/releases/2009/10/091013105324.htm]</ref>.  The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.

[[Image:13027382714469.png|thumb|280px|left|Semisynthetic RNase A. The synthetic peptide analog, RNase 111-118, is colored according to hydrophilicity. Yellow areas are comprised of hydrophobic residues. Red and brown segments are negatively and positively charged residues, respectively.]]The peptide synthesis of non-natural and non-coded proteins allowed scientists to analyze the mechanism and structure-activity relationships of classical enzyme molecules that were not accessible by traditional biomedical methods. These syntheses, though, were both difficult and time consuming, and advances in technique developed slowly<ref name = 'Merrifield'>Merrifield B. "Solid Phase Synthesis", Nobel Lecture, 8 December, 1984.</ref>. At the beginning of the twentieth century, Emil Fischer performed the first synthesis of a peptide, but it was not until 1953 that the first peptide hormone was synthesized by Du Vigneaud<ref name="Merrifield" />. The development of solid phase synthesis by Bruce Merrifield was a radical departure from traditional methods of bio-molecular synthesis that greatly increased efficiency. His method made possible the syntheses of much larger and more complex molecules; however, solid phase synthesis was not fully embraced until he demonstrated its full ability with the <scene name='Sandbox_Reserved_198/Fully_synthetic/2' target='1'>complete synthesis of Ribonuclease A</scene>. This milestone synthesis and subsequent semisynthetic syntheses of enzymes including <scene name='Sandbox_Reserved_198/Semisynthetic_rnase_a/1' target='1'>semisynthetic RNase A</scene> enriched the hypothesis that the amino acid sequence of a protein contains all necessary information to direct the formation of a fully active enzyme and, additionally, that an enzyme demonstrating the catalytic capacity and specificity of a naturally produced enzyme can be made in laboratory<ref name="Merrifield" /><ref name ='Martin'>Martin, Philip D., Marilynn S. Doscher, and Brian F. P. Edwards. "The Redefined Crystal Structure of a Fully Active Semisynthetic Ribonuclease at 1.8-A Resolution." The Journal of Biological Chemistry 262.33 (1987): 15930-5938.</ref><ref name ='Boerema'>David J. Boerema, Valentina. A. T., Stephen B. H. Kent, "Total Synthesis by Modern chemical Ligation Methods and High Resolution (1.1-A) X-ray structure of Ribonuclease A. Biopolymers. 2008;90(3):278-86.</ref>.  

The semi-synthetic RNase A comprises of residues 1-118 and the synthetic analog of residues 111-124. The RNase 1-118 was prepared by successive digestion of RNase A pepsin and carboxypeptidase A<ref>Marilynn S. Doscher, Philip D. Martin and Brian F.P. Edwards, "Characerization of the Histidine Proton Nuclear Magnetic Resonance of a Semisynthetic Ribonuclease." Biochemistry, 1983,22,4125-4131</ref>. The synthetic component, RNase 111-124, was prepared by the use of solid-phase peptide synthetic methods, in which the peptide chain was assembled in the stepwise manner while it was attached at one end to a solid support. The peptide chain was extended by repetitive steps of de-protection, neutralization and coupling until the desired sequence was obtained<ref>Lin, M. C. (1970) Journal of Biological Chemistry, 245, 6726-6731</ref>. It was important that the synthesis proceeds rapidly and in high yields to prevent side reactions or by-products.