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

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The National Academy of Sciences

RESUMO

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 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 19F-NMR, each method showing multiple kinetic phases. The 19F-NMR stopped-flow spectra, collected at 70 μM 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.

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