Stability and Folding Kinetics of Ribonuclease T 1 are Strongly Altered by the Replacement of Cis-proline 39 with Alanine

Abstract

The refolding of ribonuclease T 1 involves two major slow processes that exhibit properties of prolyl isomerization reactions. A comparison of the wild-type protein and a designed variant where the cis Ser54-Pro55 bond was replaced by a Gly54-Asn55 bond indicated that the faster of these reactions is the isomerization of Pro55. Here we report the replacement of the other cis proline of ribonuclease T 1 at position 39 by alanine. The Pro39Ala variant is similar to the wild-type protein in secondary and tertiary structure, and the enzymatic activity towards RNA and a dinucleotide substrate remains almost unchanged. The fluorescence emission of the single Trp59 is lowered by the Pro39Ala substitution, probably because Trp59 is in close contact to Pro39 in wild-type ribonuclease T 1. Unlike the substitution of cis Pro55, the Pro39Ala mutation is strongly destabilizing and reduces the Gibbs free energy of the folded protein by about 20 kJ/mol. Pro39 is buried in native RNase T 1 and located near the active site. The observed destabilization could originate from the presence of a cis alanyl bond in the Pro39Ala variant or from a local distortion caused by the incorporation of a trans alanyl peptide bond in the interior of the protein. In the refolding kinetics the replacement of Pro39 leads to a disappearance of the fast-refolding species. Refolding still involves two consecutive slow steps. The first and faster step could be the isomerization of the remaining cis Pro55. The second, very slow step is a novel reaction that appears to have no counterpart in the refolding of the wild-type protein. All mutant molecules must undergo this reaction before reaching the native state. These major changes in the folding kinetics strongly indicate that cis-Pro39 is indeed of major importance for the folding of the wild-type protein. They indicate, moreover, that some new feature of protein folding kinetics is observed in these studies of the Pro39Ala variant.