title: Conformationally driven protease-catalyzed splicing of peptide segments: V8 protease-mediated synthesis of fragments derived from thermolysin and ribonuclease A.
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 degrees 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 non-interacting complementary fragments (TC295-302 and TC303-316) in the presence of the above cosolvents at pH 6 and 4 degrees 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 beta-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.
authors.affiliation: National Institute of Immunology, New Delhi, India.
authors.forename: S
authors.initials: S
authors.lastname: Kumaran
authors.forename: D
authors.initials: D
authors.lastname: Datta
authors.forename: R P
authors.initials: RP
authors.lastname: Roy
mesh_terms: D000433:1-Propanol; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012259:Ribonuclease, Pancreatic / Q000138:chemical synthesis*; D012697:Serine Endopeptidases / Q000378:metabolism*; D012996:Solutions; D013696:Temperature; D013820:Thermolysin / Q000138:chemical synthesis*
chemical_list: D010446:Peptide Fragments; D012996:Solutions; D000433:1-Propanol; D012259:Ribonuclease, Pancreatic; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D013820:Thermolysin