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

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 [sup.19]F-NMR experiments. [sup.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 [sup.19]F-NMR, each method showing multiple kinetic phases. The [sup.19]F-NMR stopped-flow spectra, collected at 70 [micro]M of protein with a fluorine cryoprobe, demonstrated that the intermediate was populated early in the folding process ( flourescence | circular dichroism | folding intermediates | P pili