Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate
Article first published online: 3 FEB 2004
Copyright © 1992 Wiley-Liss, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 12, Issue 2, pages 171–179, February 1992
How to Cite
Kiefhaber, T., Grunert, H.-P., Hahn, U. and Schmid, F. X. (1992), Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate. Proteins, 12: 171–179. doi: 10.1002/prot.340120210
- Issue published online: 3 FEB 2004
- Article first published online: 3 FEB 2004
- Manuscript Accepted: 3 JUN 1991
- Manuscript Received: 14 MAR 1991
- folding mutant;
- proline isomerization;
- folding kinetics
The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline-limited reaction that is unusually slow in the refolding of the wild-type protein. The distribution of fast- and slow-folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp-59 is in close contact to Pro-39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the transcis reisomerization of the Tyr-38–Pro-39 bond at the stage of a native-like folding intermediate. The steric hindrance is abolished either by changing Trp-59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild-type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that transcis isomerization of Pro-39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate ratelimiting steps by trapping partially folded structures.