Real-time and equilibrium (19)F-NMR studies reveal the role of domain-domain interact
 

Real-time and equilibrium (19)F-NMR studies reveal the role of domain-domain interactions in the folding of the chaperone PapD.

Related Articles Real-time and equilibrium (19)F-NMR studies reveal the role of domain-domain interactions in the folding of the chaperone PapD.

Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):709-14

Authors: Bann JG, Pinkner J, Hultgren SJ, Frieden C


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 (