Kinetics of dissociation, unfolding, and refolding of the hexameric Hfq (Y55W) mutant.

While most cellular proteins function as oligomers, the mechanisms by which they acquire quaternary structures remain poorly understood. This study analyzes experimental data regarding the dissociation, unfolding, and refolding kinetics of the hexameric Hfq (Y55W) mutant. The dissociation of the Hfq (Y55W) quaternary structure exhibits significant sensitivity to environmental conditions. At pH 7.6 and 1.6 M guanidine hydrochloride (Gu-HCl), dissociation induced by protein concentration jump occurs much more slowly (t1/2 ≈ 34 h) than that induced by a pH jump from pH 8.0 to 1.5 (t1/2 ≈ 0.15 h). The protein unfolding induced by the Gu-HCl concentration jump from 0 M to 5.0 M results in a unfolded monomeric state, as verified using size-exclusion chromatography and protein intrinsic fluorescence spectroscopy. The Hfq (Y55W) refolding monitored via Far-ultraviolet circular dichroism (Far UV CD) at 222 nm and tryptophan (Trp) fluorescence reveals a complex, five-stage multiphasic process. Within the stopped-flow dead time (t1/2 < 5 ms), the protein forms a state competent for specific oligomerization as it is shown using Far-UV CD; this stage does not depend on protein concentration. The actual formation of the protein hexamer occurs during the first measurable kinetic stage, as detected by Trp fluorescence; this stage is concentration-dependent. The final stages show little or no dependence on protein concentration; they likely represent internal structural adjustments as the subunits settle into their native state within the hexamer.