PROTEIN FOLDING, DYNAMICS AND FUNCTION

Deciphering the mechanism by which proteins acquire their precisely folded three-dimensional structures remains one of the most important challenges of structural biology. To solve this complex problem, we need to understand not only the forces that stabilize the native structure, but also the sequence of conformational events occurring during folding; this process requires detailed kinetic studies and structural characterization of intermediate states. The protein-folding problem plays a key role in designing proteins with new or altered functional properties and improved methods for protein structure prediction. Moreover, a thorough understanding of the principles that govern folding of proteins in vitro is a prerequisite for elucidating protein folding in the cell, which has far-reaching biological and medical implications.

Much of our recent efforts have been aimed at capturing the very early stages of protein folding, which are critical to understanding how folding is directed along productive channels toward the native structure (1). We use optical and NMR methods for detailed structural, thermo-dynamic and kinetic studies on several small proteins with diverse structural characteristics including cytochrome c (cyt c), ubiquitin, staphylococcal nuclease and the B1 domain of protein G. To monitor their folding or unfolding reactions, we rely on various optical probes, such as absorbance, fluorescence and circular dichroism, coupled with rapid mixing techniques (2), including a new continuous-flow method with greatly improved time resolution (1). Hydrogen exchange and NMR methods provide insight into the structural and dynamic properties of kinetic intermediates and partially folded equilibrium states. By combining these biophysical approaches with site-specific mutagenesis, we can elucidate the role of individual residues in stabilizing the various conformational states that participate in folding. We also make use of heteronuclear multidimensional NMR methods for structural studies on proteins and peptides of biomedical interest, and model proteins resulting from de novo design efforts (3).

The goal of this project, using the horse cyt c as a test case, is to investigate the hypotheses that evolutionarily conserved interactions within the core of globular proteins are formed at an early stage of folding, and that they play a critical role in facilitating rapid and efficient acquisition of the native structure. In addition to a few residues required for heme attachment and ligation, the only strictly conserved residues among ~150 known cyt c sequences are Gly6, Phe10, Leu94 and Tyr97, which occupy central positions at the interface between the N and C-terminal helices (Figure 1a). We prepared a series of cyt c variants with mutations at these and several other key positions using a novel E. coli expression system that relies on co-expression of the enzyme heme lyase for covalent attachment of the heme group. To simplify the folding behavior, we replaced His33, which is known to form transient non-native interactions with the heme iron during folding, with Asn.

To determine the effect of mutations on protein stability, we measured the unfolding equilibrium of each variant as a function of denaturant (guanidine HCl and urea) concentration, using various optical probes. In contrast to some of the more peripheral positions, amino acid changes at helical contact sites (Phe10, Leu64, Leu94 and Tyr97) are highly destabilizing, by as much as 4.4 kcal/mol under conditions where the wild type protein has a stability of 8.2 kcal/mol. Particularly interesting are the mutations at Phe10 and Tyr97, which are involved in a phylogenetically conserved aromatic ring-ring interaction at the interface between the N- and Cterminal helices. In contrast to wild type cyt c and all other variants studied, F10I and Y97V show evidence of a well-populated equilibrium intermediate (Figure 1b). Their unfolding behavior is inconsistent with a simple two-state equilibrium, but can be modeled quantitatively in terms of a three-state folding mechanism, N ´ I ´ U. The intermediate, I, can be distinguished spectroscopically from both the native and the unfolded states, N and U. N ´ I is a major cooperative unfolding transition characterized by a strongly denaturant-dependent equilibrium constant, and I ´ U is a less cooperative transition characterized by a shallow transition. Although the order of the two transitions cannot be determined from equilibrium measurements alone, the equilibrium intermediate appears to be closely related to an early kinetic intermediate, based on similarities in the midpoint of the transition and dependence on denaturant concentration. Surprisingly, the equilibrium intermediates are well populated only for F10I and Y97V, but not for the other destabilizing mutations such as L94A or L64A. This may be due to the fact that aliphatic substitutions at positions 10 or 97 replace a specific aromatic ring-ring interaction with a less directional hydrophobic interaction, which has little effect on the loosely packed helix-helix pair in the intermediate, but strongly destabilizes the tightly packed native state. Thus, disruption of the aromatic contact has little effect on the U<->I equilibrium, but raises the free energy of N relative to I and U such that I becomes detectable at equilibrium.

Insight into the structural properties of the intermediate was obtained by comparing the equilibrium behavior of the F10I and Y97V mutants for various spectroscopic probes (Figure 1c), including absorbance in the Soret region to monitor changes in heme ligation and environment, far-UV CD to monitor changes in (helical) secondary structure, near-UV CD to monitor more specific tertiary interactions among aromatic groups, and fluorescence of Trp59, which provides a qualitative measure of chain dimensions. The results indicate that the equilibrium intermediate in the denaturant-induced unfolding transition of the F10I and Y97V variants is compact and contains extensive secondary structure, but lacks the native Met80 heme ligation and specific tertiary interactions characteristic of the native cyt c structure. The unusual unfolding behavior of these mutants indicates that replacement of the specific aromatic ring-ring interaction with a regular hydrophobic contact selectively destabilizes the native structure while allowing accumulation of a partially structured intermediate, which has previously been detected only as a transient kinetic intermediate.

EARLY EVENTS IN FOLDING OF CYTOCHROME C. SHASTRY, RODER

For many proteins, time-resolved measurements of refolding using stopped-flow methods show evidence for rapid changes in the optical signal monitored (e.g., intrinsic or extrinsic fluorescence probes or far-UV circular dichroism) within the instrumental dead time (>1 ms). This so-called "burst phase effect" is often attributed to the rapid accumulation of partially folded intermediates in a reversible transition preceding the rate-limiting step in folding. Alternatively, the results can be explained in terms of a model involving a solvent-dependent redistribution of denatured conformations. While formation of an intermediate gives rise to exponential kinetics, the continuous contraction of the polypeptide chain invoked by the latter model would be characterized by a non-exponential time course. In order to discriminate between these possibilities, it is necessary to directly observe the kinetics of folding on the sub-millisecond time scale. These observations were made possible by our recent development of a new continuous-flow capillary mixing apparatus that can resolve fluorescence changes down to the 10 ms range. Our previous studies on horse cyt c showed that the initial collapse of the polypeptide chain during refolding is indeed limited by a kinetic barrier rather than chain diffusion (1).

In a series of continuous-flow folding measurements under a variety of initial (acid- or solvent-induced unfolded states) and final conditions (native state at neutral or mildly acidic pH, imidazole complex, etc.), we consistently observed a prominent exponential decay in fluorescence with a time constant of 30-65 ms. This suggests that the first kinetic barrier encountered during folding is largely entropic in nature and may represent a general bottleneck caused by an incomplete compensation of unfavorable entropic contributions from favorable intramolecular and solvent interactions. We recently observed a similar event under acidic conditions favoring formation of a partly folded equilibrium state (the Astate), which has some of the characteristics of a molten globule. Formation of the cyt c A-state is coupled with binding of a chloride ion and can be induced by adding salt to the acid-unfolded state at pH 2. At high salt concentrations (1 M KCl), the continuous-flow fluorescence measurements exhibit a single-exponential decay with a time constant of 50 ms indicating formation of a compact intermediate similar to that observed under native conditions. Conversion of this intermediate into the A-state gives rise to an

additional process with a time constant of ~1 ms that appears at lower salt concentrations. Thus, similar conformational barriers and intermediates are encountered both under native conditions and during formation of the A-state, indicating that the latter represents a late folding intermediate.

Many small proteins can fold in a single concerted step without detectable intermediates. One notable exception is the B1 domain of streptococcal protein G (GB1). Despite its small size, 57 residues, and simple architecture, consisting of a four-strand b-sheet and a flanking a-helix (Figure 2), GB1 exhibits a pronounced burst phase in stopped-flow fluorescence measurements under strongly native conditions (Park et al., Biochemistry 47:14277, 1997). Using our continuous-flow capillary mixing device, we recently extend these measurements down to the 100 ms range, which allowed us to observe the previously unresolved fluorescence changes directly. The refolding reaction, initiated by a rapid drop in denaturant concentration, was monitored by measuring the increase in the fluorescence emission associated with the burial of a tryptophan side chain (Trp 43) within the hydrophobic core of GB1.

Figure 2 shows the kinetic traces obtained in a series of continuous-flow experiments at different final denaturant concentrations, along with stopped-flow traces obtained under matching conditions. Under strongly stabilizing conditions, the kinetics are dominated by an exponential increase in fluorescence with a time constant of 600 ± 20 ms, followed by a minor phase in the millisecond range. With increasing denaturant concentration, the rate-limiting process on the stopped-flow time scale slows down and gains amplitude at the expense of the fast process, whose rate remains nearly constant. The combined kinetic traces quantitatively account for the total difference in fluorescence between the native and unfolded state at equilibrium, including the previously unresolved "burst phase" signal. Thus, our observations capture the complete time course of folding observable by fluorescence, which measures the transfer of Trp43 from an aqueous to an apolar environment and concomitant formation of the hydrophobic core.

Kinetic simulations indicate that the denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state folding mechanism, U <-> I <-> N, where I is an obligatory folding intermediate on a direct path between the unfolded and native states. The observation of an exponential time course during the initial stages of folding confirms that the formation of I from U is a barrier-crossing event. In addition, the fact that the fluorescence signal, after the initial phase approaches the level reached of the native state indicates that Trp43 is already sequestered in I within a largely solvent-shielded environment. It is interesting that this compact intermediate is formed at a ~10-fold slower rate in GB1 compared to cyt c. Given that cyt c is nearly twice as large as GB1, protein size is apparently not an important factor in determining the rate of early folding events. However, this surprising result may well be related to the fact that GB1 contains extensive b-sheet structure, including a pair of parallel strands involving opposite ends of the chain, which is expected to be a slower process than coalescence of a-helices in proteins such as cyt c.

PUBLICATIONS

1.   SHASTRY, M.C.R., SAUDER, J.M., RODER, H. Kinetic and structural analysis of submillisecond folding events in cytochrome c. Accts. Chem. Res. 31:717-725, 1998.

2.   RANKIN, S.E., WATTS, A., RODER, H., PINHEIRO, T.J.T. Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state. Protein Sci. 8:381-393, 1999.

3.   WALSH, S.T.R., CHENG, H., BRYSON, J.W., RODER, H., DEGRADO, W.F. Solution structure and dynamics of a de novo designed three helix bundle protein. Proc. Natl. Acad. Sci. USA 96:5486-5491, 1999.

4.   DOLGIKH, D.A., LATYPOV, R.F., ABDULLAEV, Z.K., COLÓN, W., RODER, H., KIRPICHNIKOV, M.P. Expression of mutant horse cytochrome c genes in Escherichia coli. Bioorg. Khim. 24:756-759, 1998.

a    S.-H. Park:  Present address--Division of Protein Engineering, KRIBB, KIST, Taejon 305-600, S. Korea

b    O.B. Ptitsyn (deceased): Laboratory of Mathematical Biology, NIH, Rockville, MD 20850

c    D.A. Dolgikh: Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998