Conformationally driven protease-catalyzed splicing of peptide segments: V8 protease-mediated synthesis of fragments derived from thermolysin and ribonuclease A
✍ Scribed by Sangaralingam Kumaran; Debjani Datta; Rajendra P. Roy
- Book ID
- 105356484
- Publisher
- Cold Spring Harbor Laboratory Press
- Year
- 2008
- Tongue
- English
- Weight
- 864 KB
- Volume
- 6
- Category
- Article
- ISSN
- 0961-8368
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✦ Synopsis
Abstract
We have studied the conformation as well as V8 protease‐mediated synthesis of peptide fragments, namely amino acid residues 295‐316 (TC‐peptide) of thermolysin and residues 1‐20 (S‐peptide) of ribonuclease A, to examine whether “conformational trapping” of the product can facilitate reverse proteolysis. The circular dichroism study showed cosolvent‐mediated cooperative helix formation in TC‐peptide with attainment of about 30‐35% helicity in the presence of 40% 1‐propanol and 2‐propanol solutions at pH 6 and 4°C. The thermal melting profiles of TC‐peptide in the above cosolvents were very similar. V8 protease catalyzed the synthesis of TC‐peptide from a 1:1 mixture of the noninteracting complementary fragments (TC295‐302 and TC303‐316) in the presence of the above cosolvents at pH 6 and 4°C. In contrast, V8 protease did not catalyze the ligation of S1‐9 and S10‐20, although S‐peptide could assume helical conformation in the presence of the cosolvent used for the semisynthetic reaction. V8 protease was able to synthesize an analog of S‐peptide (SA‐peptide) in which residues 10‐14 were substituted (RQHMD → VAAAK). While S‐peptide exhibited helical conformation in the presence of aqueous propanol solutions, SA‐peptide displayed predominantly β‐sheet conformation. SA‐peptide showed enhanced resistance to proteolysis as compared with S‐peptide. Thus, failure of semisynthesis of S‐peptide may be a consequence of high flexibility around the 9‐10 peptide bond due to its proximity to the helix stop signal. The results suggest that protease‐mediated ligations may be achieved by design and manipulation of the conformational aspects of the product.