The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S-peptide analogues with enhanced helical stability
✍ Scribed by Dr. Colin Mitchinson; Robert L. Baldwin
- Book ID
- 105358358
- Publisher
- John Wiley and Sons
- Year
- 1986
- Tongue
- English
- Weight
- 938 KB
- Volume
- 1
- Category
- Article
- ISSN
- 0887-3585
No coin nor oath required. For personal study only.
✦ Synopsis
Recent work has shown that, with synthetic analogues of C-peptide (residues 1-13 of ribonuclease A), the stability of the peptide helix in H2O 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 Tm of the reconstituted ribonuclease S. Six peptides have been made, which contain Glu9----Leu, a blocked alpha-COO- group (-CONH2), 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 Tm compared to the protein with S-peptide (1-19). The Tm increases as the charge on residue 1 changes from +2 to -1. The largest increment in Tm is 6 degrees. The results suggest that the stability of a protein can be increased by enhancing the stability of its secondary structure.