The slow folding 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-Cys10 disulfide bond, adjoining the original termini with a three-peptide Gly-Gly-Gly linker, and conferring new termini to four different solvent-exposed beta-turns interposing secondary structural elements [Garrett, J. B., Mullins, L. S., & Raushel, F. M. (1996) Protein Sci. 5, 204-211]. Each of the mutant proteins continues to be rate-limited in folding by the slow trans to cis isomerizations of Pro39 and Pro55, giving rise to a branched mechanism populated by intermediates with mixed proline isomers. However, the overall rate of folding is increased in accordance with the general destabilizing effect of each circular permutation. Steric hindrances imposed by Trp59 on the isomerization around the Tyr38-Pro39 peptide bond have been implicated in decelerating the folding of RNase T1 [Kiefhaber, T., Grunert, H.-P., Hahn, U., & Schmid, F. X. (1992) Proteins: Struct., Funct., Genet. 12, 171-179]; it is this tertiary restraint which appears to be variably relieved by the sequence transpositions. A fluorescence characterization of Trp59 indicates little difference between fully folded RNase T1 and the variants in terms of its lifetime, accessibility to quenchers, and rotational properties. Yet, within protein that is "completely" denatured, Trp59 exhibits variable flexibility, greatest within the circularly permuted variants folding 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. Study holds ProTherm entries: 4826, 4827 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:27 p.m.
|Number of data points||6|
|Proteins||Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence ACDYTCGSNCYSSSDVSTAQAAGYQLHEDGETVGSNSYPHKYNNYEGFDFSVSSPYYEWPILSSGDVYSGGSPGADRVVFNENNQLAGVITHTGASGNNFVECT|