title: Real-time and equilibrium (19)F-NMR studies reveal the role of domain-domain interactions in the folding of the chaperone PapD.
abstract: PapD is a periplasmic chaperone essential for P pilus formation in pyelonephritic strains of E. coli. It is composed of two domains, each of which contains a tryptophan residue (Trp-36 and Trp-128, in the N- and C-terminal domains, respectively). To explore the role of domain-domain interactions during folding, the protein was labeled with 6-fluorotryptophan for use in (19)F-NMR experiments. (19)F-NMR data collected as a function of urea concentration revealed the presence of a resonance caused by Trp-128 that was distinct from either the folded or unfolded resonances. The time course of refolding from urea was monitored by stopped-flow fluorescence, CD, and (19)F-NMR, each method showing multiple kinetic phases. The (19)F-NMR stopped-flow spectra, collected at 70 microM of protein with a fluorine cryoprobe, demonstrated that the intermediate was populated early in the folding process (<5 s). The slow disappearance of the intermediate and unfolded resonance occurred at the same rate as the appearance of the native resonances of both domains. The data are consistent with a model in which the C-terminal domain collapses rapidly to an intermediate, whereas the stabilization of the final structure is slow and requires folding of the N-terminal domain with concomitant readjustment of the C-terminal domain structure.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: James G
authors.initials: JG
authors.lastname: Bann
authors.forename: Jerome
authors.initials: J
authors.lastname: Pinkner
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Hultgren
authors.forename: Carl
authors.initials: C
authors.lastname: Frieden
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005461:Fluorine / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D033903:Periplasmic Proteins; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry; D014508:Urea
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C084330:PapD protein, E coli; D033903:Periplasmic Proteins; D005461:Fluorine; D014364:Tryptophan; D014508:Urea