Observation of Multistate Kinetics during the Slow Folding and Unfolding of Barstar

Abstract

The kinetics of the slow folding and unfolding reactions of barstar, a bacterial ribonuclease inhibitor protein, have been studied at 23(+/-1) degrees C, pH 8, by the use of tryptophan fluorescence, far-UV circular dichroism (CD), near-UV CD, and transient mixing (1)H nuclear magnetic resonance (NMR) spectroscopic measurements in the 0-4 M range of guanidine hydrochloride (GdnHCl) concentration. The denaturant dependences of the rates of folding and unfolding processes, and of the initial and final values of optical signals associated with these kinetic processes, have been determined for each of the four probes of measurement. Values determined for rates as well as amplitudes are shown to be very much probe dependent. Significant differences in the intensities and rates of appearance and disappearance of several resolved resonances in the real-time one-dimensional NMR spectra have been noted. The NMR spectra also show increasing dispersion of chemical shifts during the slow phase of refolding. The denaturant dependences of rates display characteristic folding chevrons with distinct rollovers under strongly native as well as strongly unfolding conditions. Analyses of the data and comparison of the results obtained with different probes of measurement appear to indicate the accumulation of a myriad of intermediates on parallel folding and unfolding pathways, and suggest the existence of an ensemble of transition states. The energetic stabilities of the intermediates estimated from kinetic data suggest that they are approximately half as stable as the fully folded protein. The slowness of the folding and unfolding processes (tau = 10-333 s) and values of 20.5 (+/-1.4) and 18 (+/-0.5) kcal mol(-)(1) for the activation energies of the slow refolding and unfolding reactions suggest that proline isomerization is involved in these reactions, and that the intermediates accumulate and are therefore detectable because the slow proline isomerization reaction serves as a kinetic trap during folding.

Identification and Characterization of an Equilibrium Intermediate in the Unfolding Pathway of an All β-Barrel Protein

“…Interestingly, the unfolding of the protein in 2 M GdnHCl is very slow, and it takes more than 20 min for complete unfolding of the protein. The unfolding and refolding rates were measured as a function of the denaturant (GdnHCl) concentration to produce a classical Chevron plot (36,37). The transition midpoint is observed around 1.3 M and agrees closely with that estimated from the equilibrium unfolding data (Fig.…”

Exploring the Cooperativity of the Fast Folding Reaction of a Small Protein Using Pulsed Thiol Labeling and Mass Spectrometry

“…A rapid equilibrium between U and I E is established before the major structural transition to the late intermediate I L occurs. I L has also been shown to be a heterogeneous ensemble of intermediates (32)(33)(34). Different members of the I E ensemble are populated in different solvent conditions; hence, the structural properties of I E appear different in different folding conditions (30,31).…”

Osmolytes Induce Structure in an Early Intermediate on the Folding Pathway of Barstar

“…More recent studies indicate that I E is an ensemble of at least three different structural forms, each of which is stabilized differentially, and hence populated differentially, in the absence and presence of salts (20). I L also shows structural heterogeneity (34). Most recently, the use of a multisite time-resolved fluorescence resonance energy transfer approach has shown that the extent of structural heterogeneity depends on how stable the folding conditions are and that different structural components predominate in I L under different folding conditions (35).…”