title: Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A.
abstract: The earliest folding events in single-tryptophan mutants of RNase A were investigated by fluorescence measurements by using a combination of stopped-flow and continuous-flow mixing experiments covering the time range from 70 micros to 10 s. An ultrarapid double-jump mixing protocol was used to study refolding from an unfolded ensemble containing only native proline isomers. The continuous-flow measurements revealed a series of kinetic events on the submillisecond time scale that account for the burst-phase signal observed in previous stopped-flow experiments. An initial increase in fluorescence within the 70-micros dead time of the continuous-flow experiment is consistent with a relatively nonspecific collapse of the polypeptide chain whereas a subsequent decrease in fluorescence with a time constant of approximately 80 micros is indicative of a more specific structural event. These rapid conformational changes are not observed if RNase A is allowed to equilibrate under denaturing conditions, resulting in formation of nonnative proline isomers. Thus, contrary to previous expectations, the isomerization state of proline peptide bonds can have a major impact on the structural events during early stages of folding.
authors.affiliation: Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301, USA.
authors.forename: Ervin
authors.initials: E
authors.lastname: Welker
authors.forename: Kosuke
authors.initials: K
authors.lastname: Maki
authors.forename: M C Ramachandra
authors.initials: MC
authors.lastname: Shastry
authors.forename: Darmawi
authors.initials: D
authors.lastname: Juminaga
authors.forename: Rajiv
authors.initials: R
authors.lastname: Bhat
authors.forename: Harold A
authors.initials: HA
authors.lastname: Scheraga
authors.forename: Heinrich
authors.initials: H
authors.lastname: Roder
mesh_terms: D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D014443:Tyrosine; D012259:Ribonuclease, Pancreatic