Sandbox Reserved 198

Fully Synthetic</scene>

Semisynthetic Ribonuclease A

2-D Semisynthetic RNase A

Introduction

The synthesis of proteins allowed scientists to analyze biological molecules through manipulations that could not readily be made with natural proteins. These syntheses, though, were very difficult, required large investments of time, and advances in technique did not occur frequently. 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. 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 complete synthetic synthesis of Ribonuclease A. This milestone synthesis and subsequent semisynthetic syntheses of enzymes including RNase A 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.

Function

The structure to function relationship is clearly exhibited by semisynthetic RNase A. In the RNase A protein, the removal of six C terminal residues, leaving , completely halts enzymatic activity (Martin, 1987). However, a complex of RNase 1-118 with a synthetic polypeptide comprising the restores enzymatic activity to RNase A. Upon the addition of the synthetic chain, the semisynthetic enzyme adopts a structure that closely resembles that of natural RNase (Martin, 1987). The restoration of the structure reconstitutes the enzymatic activity of RNase to 98% (Martin, 1987).

Synthetic Method

The RNase 1-118 was prepared by successive digestion of RNase A pepsin and carboxypeptidase A (Doscher, 1983). The synthetic component, RNase 111-124, was prepared by the use of solid-phase peptide synthetic mothods, in which the peptide chain was assembled in the stepwise mannar while it was attached at one end to a solid support. The peptide chain was extented by repetitive steps of deprotection, neutralization and coupling until the desired sequence was obtained (Lin, 1970). It was important that the synthesis proceeds rapidly and in high yields to prevent side reactions or by-products.

Related Web-links

 1. Introduction to Ribonuclease A by Raines: http://www.uta.edu/faculty/sawasthi/Enzymology-4351-5324/Class%20Syllabus%20Enzymology/ribonucleaseA.pdf
 2. Solid Phase Synthesis by Merrifield (Nobel Prize Winner):http://nobelprize.org/nobel_prizes/chemistry/laureates/1984/merrifield-lecture.pdf
 3. Chemical Synthesis of Proteins:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845543/?tool=pmcentrez
 4. Refined Crystal Structure: http://www.ncbi.nlm.nih.gov/pubmed/3680234

References

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.

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.

Lin, M. C. (1970) Journal of Biological Chemistry, 245, 6726-6731.

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.

Link title