ARE TURNS REQUIRED FOR THE FOLDING OF RIBONUCLEASE-T1

Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino a cids for which extensive thermodynamic and structural information is k nown. To assess the specific influence of variations in amino acid seq uence on the mechanism for protein folding, circularly permuted varian ts of RNase T1 were constructed and characterized in terms of catalyti c activity and thermodynamic stability. The disulfide bond connecting Cys-2 and Cys-10 was removed by mutation of these residues to alanine (C2, 10A) to avoid potential steric problems imposed by the circular p ermutations. The original amino-terminus and carboxyl-terminus of the mutant (C2,10A) were subsequently joined with a tripeptide linker to a ccommodate a reverse turn and new termini were introduced throughout t he primary sequence in regions of solvent-exposed loops at Ser-35 (cp3 5S1), Asp-49 (cp49D1), Gly-70 (cp70G1), and Ser-96 (cp96S1). These cir cularly permuted RNase T1 mutants retained 35-100% of the original cat alytic activity for the hydrolysis of guanylyl(3' --> 5') cytidine, su ggesting that the overall tertiary fold of these mutants is very simil ar to that of wild-type protein. Chemical denaturation curves indicate d thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/m ol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild-type RNase T1 and 6.4 kcal/mol for (C 2, 10A) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight beta-turn between Glu-82 and Gl n-85. New termini were inserted at Asn-83 (cp83N1), Asn-84 (cp84N1), a nd Gln-85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1.