title: Examination of the folding of E. coli CspA through tryptophan substitutions.
abstract: Escherichia coli cold shock protein, CspA, folds very rapidly (time constant, tau = 4 msec) by an apparent two-state mechanism. However, recent time-resolved infrared (IR) temperature-jump experiments indicate that the folding trajectory of CspA may be more complicated. The sole tryptophan of wild-type CspA (Trp11), which is used to monitor the folding process by fluorescence spectroscopy, is located in an unusual aromatic cluster on the surface of CspA within the nucleic acid binding site. To gain a more global picture of the folding kinetics of CspA and to determine if there are any previously undetected intermediates, we have introduced a second tryptophan at three different surface locations in the protein. The three mutations did not significantly alter the tertiary structure of CspA, although two of the substitutions were found to be slightly stabilizing. Two-state folding, as detected by stopped-flow fluorescence spectroscopy, is preserved in all three mutants. These results indicate that the fast folding of CspA is driven by a concerted mechanism.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Vu
authors.forename: K L
authors.initials: KL
authors.lastname: Reid
authors.forename: H M
authors.initials: HM
authors.lastname: Rodriguez
authors.forename: L M
authors.initials: LM
authors.lastname: Gregoret
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C062260:cold shock protein CS7.4, Bacteria; D014364:Tryptophan