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The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S‐peptide analogues with enhanced helical stability
Author(s) -
Mitchinson Colin,
Baldwin Robert L.
Publication year - 1986
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340010106
Subject(s) - peptide , ribonuclease , thermostability , residue (chemistry) , chemistry , helix (gastropod) , circular dichroism , thermal stability , stereochemistry , biochemistry , enzyme , biology , rna , organic chemistry , ecology , snail , gene
Recent work has shown that, with synthetic analogues of C‐peptide (residues 1–13 of ribonuclease A), the stability of the peptide helix in H 2 O depends strongly on the charge on the N‐terminal residue. We have asked whether, in semisynthetic ribonuclease S reconstituted from S‐protein plus an analogue of S‐peptide (1–15), the stability of the peptide helix is correlated with the T m of the reconstituted ribonuclease S. Six peptides have been made, which contain Glu9 → Leu, a blocked α‐COO − group (CONH 2 ), and either Gln11 or Glu11. The N‐terminal residue has been varied; its charge varies from +2 (Lys) to −1 (succinyl‐Ala). We have measured the stability of the peptide helix, the affinity of the peptide for S‐protein (by C.D. titration), and the thermal stability of the reconstituted ribonuclease S. All six peptide analogues show strongly enhanced helix formation compared to either S‐peptide (1–15) or (1–19), and the helix content increases as the charge on the N‐terminal residue changes from +2 to −1. All six peptides show increased affinity for S‐protein compared to S‐peptide (1–19), and all six reconstituted ribonucleases S show an increase in T m compared to the protein with S‐peptide (1–19). The T m increases as the charge on residue 1 changes from +2 to −1. The largest increment in T m is 6°. The results suggest that the stability of a protein can be increased by enhancing the stability of its secondary structure.