Sandbox Reserved 198: Difference between revisions

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==Introduction==

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>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>Merrifield B. "Solid Phase Synthesis", Nobel Lecture, 8 December, 1984.</ref>. 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'>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'>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>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>Merrifield B. "Solid Phase Synthesis", Nobel Lecture, 8 December, 1984.</ref><ref>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 synthesis of semisynthetic RNasa A clearly exhibits the structure to function relationship that defines proteins. In the RNase A protein, the removal of six C terminal residues, leaving <scene name='Sandbox_Reserved_198/Rnase_1-118/1'>RNase 1-118</scene>, completely halts enzymatic activity<ref>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>. However, a complex of RNase 1-118 with a synthetic polypeptide comprising the C terminal residues <scene name='Sandbox_Reserved_198/Synthetic_component/3'>111-124</scene> restores enzymatic activity to RNase A. Upon the addition of the synthetic chain, the <scene name='Sandbox_Reserved_198/Interface/7'>semisynthetic enzyme</scene> <scene name='Sandbox_Reserved_198/Interface/8'>(Zoom)</scene> adopts a structure that closely resembles that of <scene name='Sandbox_Reserved_198/Wild_type/1'>Wild Type RNase A</scene><ref>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>. The restoration of the structure reconstitutes the enzymatic activity of RNase to 98%<ref>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>.

''Fully Synthetic RNase A''  

The peptide ligation chemistry in addition to solid-phase peptide synthesis is used to synthesize relatively longer peptide molecules with typical length of 125 residues<ref>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 ligation methods overcome the length limitation of solid-phase synthesis, because the chemical ligation involves the joining of mutually reactive peptide segments created by solid-phase synthesis. The peptide bond in ligation is formed between an unprotected peptide and a peptide-thioester<ref>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 shorter peptide segments are more rapidly prepared and are less susceptible to solubility issues in longer peptide chains.  

   1. Introduction to Ribonuclease A by Raines: http://www.uta.edu/faculty/sawasthi/Enzymology-4351-5324/Class%20Syllabus%20Enzymology/ribonucleaseA.pdf

   2. Introduction to Peptide Synthesis: http://en.wikipedia.org/wiki/Solid_phase_peptide_synthesis#Solid-phase_synthesis  

   4. Chemical Synthesis of Proteins:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845543/?tool=pmcentrez

   5. Refined Crystal Structure: http://www.ncbi.nlm.nih.gov/pubmed/3680234

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