Folding pathways of circularly permuted ribonuclease T1.
Academic Article
Overview
Additional Document Info
View All
Overview
abstract
The slow refolding of circularly permuted variants of ribonuclease T1 has been examined using steady-state and frequency-domain fluorescence spectroscopy. The sequence transpositions have previously been designed by eliminating a restrictive Cys2-CyslO disulfide bond, adjoining the original termini with a 3-peptide Gly-Gly-Gly linker, and conferring new termini to four different solvent-exposed -turns interposing secondary structural elements. Each of the mutant proteins continues to be rate-limited in refolding by the slow reisomerizations from trans to cis of Pro39 and Pro55, giving rise to a branched mechanism populated by intermediates with mixed proline isomers. However, the overall rate of refolding is increased in accordance with the general destabilizing effect of each circular permutation. Steric hindrances imposed by Trp59 on the reisomerization around the Tyr38-Pro39 peptide bond have been implicated in decelerating the refolding of RNase Tl; it is this t! erti ary restraint which appears to be variably relieved by the sequence transpositions. A fluorescence characterization of Trp59 indicates little difference between fully folded RNase Tl and the variants in terms of its lifetime, accessibility to quenchers, and rotational properties. Yet, within denatured protein, Trp59 exhibits variable flexibility, greatest within the circularly permuted variants refolding the fastest. Such differences in the dynamic properties of Trp59 between each denatured protein may be direct evidence for a relative loosening of the tertiary fold maintaining the deleterious Trp59-Pro39 interaction in the partially folded intermediates.
