Circular permutations of ribonuclease T1
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abstract
To assess the specific influence of variations in ami no acid sequence on the mechanism for protein folding, circularly permuted variants of ribonuclease Tl (RNase Tl) were constructed and characterized both catalytically and thermodynamically. The disulfide bond connecting Cys-2 and Cys-10 was removed by mutation of these residues to alanine (C2,10A) thereby avoiding potential steric restraints imposed by the circular permutations. The termini of C2,10A were subsequently joined with a 3-peptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent exposed loops. Sites chosen for new termini were Ser-35 (cp35Si), Asp-49 (cp49Dl), Gly-70 (cp70Gl), and Ser96 (cp96Sl). These circularly permuted RNase Tl mutants retained 35 100% of the original catalytic activity for the hydrolysis of GpC, suggesting that the overall tertiary fold of these mutants is very similar to that of wild-type protein. Urea denaturatio n curves were used to probe thermodynamic stabilities of all the mutants. New termini were inserted at Asn-83 (cp83Nl), Asn-84 (cp84Nl), and Gin-85 (cp85Ql) but no detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn is critical for the folding and/or thermodynamic stability of RNase Tl. Further characterizations of circularly permutated proteins include incorporation of a random linker sequence in the cp35Sl mutant.
