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

摘要

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 ~19F-NMR experiments. ~19F-NMR data collected as a function of urea concentration re- vealed 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 ~19F-NMR, each method showing multiple kinetic phases.