Submillisecond protein folding kinetics studied by ultrarapid mixing

C Chan

Y Hu

S Takahashi

D Rousseau

W Eaton

J Hofrichter

^{Laboratory of Chemical Physics, Building 5, National Institutes of Health, Bethesda, MD 20892-0520; and}^{AT&T Bell Laboratories, Murray Hill, NJ 07974}

^{Present address: Department of Molecular Engineering, Kyoto University, Kyoto 606, Japan.}

^{Present address: Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.}

^{To whom reprint requests should be addressed.}

David Davies, National Institutes of Health, Bethesda, MD

Received 1996 Oct 23; Accepted 1996 Dec 5.

Abstract

An ultrarapid-mixing continuous-flow method has been developed to study submillisecond folding of chemically denatured proteins. Turbulent flow created by pumping solutions through a small gap dilutes the denaturant in tens of microseconds. We have used this method to study cytochrome c folding kinetics in the previously inaccessible time range 80 μs to 3 ms. To eliminate the heme–ligand exchange chemistry that complicates and slows the folding kinetics by trapping misfolded structures, measurements were made with the imidazole complex. Fluorescence quenching due to excitation energy transfer from the tryptophan to the heme was used to monitor the distance between these groups. The fluorescence decrease is biphasic. There is an unresolved process with τ < 50 μs, followed by a slower, exponential process with τ = 600 μs at the lowest denaturant concentration (0.2 M guanidine hydrochloride). These kinetics are interpreted as a barrier-free, partial collapse to the new equilibrium unfolded state at the lower denaturant concentration, followed by slower crossing of a free energy barrier separating the unfolded and folded states. The results raise several fundamental issues concerning the dynamics of collapse and barrier crossings in protein folding.

Keywords: cytochrome c, fluorescence, Forster energy transfer, polymer dynamics

Abstract

Under suitable conditions it would appear that most proteins refold from the chemically denatured state (1, 2). As a consequence, rapid dilution of a denaturant using the stopped-flow method has been the most useful and common experimental technique for studying protein folding kinetics (3–5). A major limitation in this method, however, is that kinetic studies are restricted to times longer than a few milliseconds. Collapse of the polypeptide chain and secondary structure formation therefore often occur before any observations can be made (6). It has been suggested, moreover, that much of the folding kinetics observed up to now correspond to the escape of partially folded or misfolded structures from traps in the energy landscape, revealing little about the fast direct pathways to the native state (7, 8).

Observation of submillisecond processes in protein folding has presented a challenge that has stimulated considerable interest in developing new experimental methods. Dramatic improvement in time resolution has been obtained by using short laser pulses to initiate folding, either by photochemical triggering (9–13) or by temperature-jump (14–17). The results of these studies are discussed in two recent reviews (18, 19) in terms of new theoretical developments in understanding how proteins fold (8, 20–23). To observe submillisecond folding kinetics we have developed an ultrarapid continuous-flow mixing method. The method has previously been used to study very fast bimolecular reactions (24–26). The critical feature for achieving rapid mixing is that the solutions are pumped through a very narrow gap, resulting in high shear that creates turbulence (24). Turbulence “breaks” the liquids into very small volume elements (27). Mixing is rapid because diffusion now occurs over short distances (∼0.1 μm). Here we report our initial results with this method, where folding of cytochrome c is monitored by excitation energy transfer from tryptophan to the heme.

We chose cytochrome c for our initial studies with this new technique because there is a very large submillisecond fluorescence change upon folding (7, 28–31) and the physics of this spectroscopic change can be reasonably well understood. There is a single tryptophan in horse cytochrome c, ∼40 residues distant along the 104 amino acid sequence from the covalently connected heme group. In the chemically denatured state, there is partial quenching of tryptophan fluorescence from excitation energy transfer to the heme. In the native structure the heme-to-tryptophan distance is so short (∼1 nm) that the tryptophan fluorescence is almost completely quenched. This fluorescence is therefore a sensitive probe for measuring the decrease in heme–tryptophan distance, which is presumed to reflect the overall collapse of the unfolded protein (7, 28–31).

To eliminate the heme ligand-exchange chemistry that complicates and slows the kinetics by trapping misfolded structures (7, 29–31), even at acid pH (32), we have studied folding with the extrinsic ligand imidazole bound to the heme (29, 33). Imidazole binding prevents intramolecular ligation of the heme by histidines in positions 26 and 33 in the unfolded state (34); it dissociates much more slowly than folding (29), and it remains bound in place of methionine in the folded structure (35, 36). In this way folding of cytochrome c more nearly resembles that of a protein that has no prosthetic group.

Acknowledgments

We thank Attila Szabo and Peter Wolynes for many helpful discussions, Garrott Christoph for the preparation of purified apomyoglobin, and Ettore Appella for the synthesis and purification of the nonapeptide. S.T. and D.L.R. acknowledge support of National Institutes of Health Grant GM-48714.

Acknowledgments

ABBREVIATION

Gdn·HCl

guanidine hydrochloride

ABBREVIATION

Footnotes

^{Our 266-nm laser light also excites tyrosines, which transfer energy to both the heme and the tryptophan. We estimate that interference from excitation of the four tyrosines is small, but it is nevertheless a complication and the observed quantum yield may overestimate the heme–tryptophan distance.}

^{We should point out that in no case has the analysis been rigorous. Strict two-state behavior has not been demonstrated in any of these studies, including our own, nor is it expected because of the complication introduced by proline isomerization. Further complications include the absence of a clear linear region in the pH 5 kinetic data (}31) and the nonexponential time course observed for folding of reduced cytochrome c (11, 12). There are 4 trans prolines in the native conformation of cytochrome. If we assume that the cis/trans ratio in the denatured state is 0.2, the fraction of all-trans conformers in the unfolded state is about (5/6)^{(∼50%). Upon dilution of Gdn·HCl, all of the tryptophan fluorescence is observed to decay in a single relaxation. From this result, we tentatively conclude that all unfolded conformers pass over free energy barriers of similar height to reach either the native state or highly compact near-native states in which the fluorescence is almost completely quenched and from which the prolines isomerize slowly to produce the native state. The apparent equilibrium constant obtained from a two-state analysis of kinetic data is not equal to the equilibrium constant for folding, because the prolines have sufficient time to isomerize in the equilibrium experiment. Nonetheless, if it is assumed that the distribution of proline isomers in both the native and denatured states is independent of Gdn·HCl concentration, then the apparent and true equilibrium constants differ by a constant Gdn·HCl-independent factor and the slope of the linear free energy plot is not affected by proline isomerization. In carrying out a simultaneous fit of the kinetic and equilibrium data, however, [native]/[unfolded] obtained from the equilibrium curve must be scaled by the factor 1 + Σ[}cis isomers]/[all-trans isomer], for the unfolded state, before using it to calculate the ratio of the folding to the unfolding rates.

Footnotes

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