PROTEIN FOLDING, DYNAMICS, AND FUNCTION

Much of our research continues to center around the protein folding problem. Structural and kinetic information on folding events and structural intermediates is essential for understanding the mechanism of folding and the interactions involved in directing the polypeptide chain toward its native fold. We are using a variety of experimental approaches to investigate the kinetics and energetics of folding for several well-characterized proteins, including cytochrome c, ubiquitin, and staphylococcal nuclease, as well as natural and engineered variants of these proteins. For the study of protein structure and dynamics, we rely on multidimensional nuclear magnetic resonance (NMR) spectroscopy, often in conjunction with hydrogen exchange methods. To monitor the kinetics of folding or unfolding reactions, we use various optical probes, such as absorbance, static and time-resolved fluorescence, and circular dichroism, coupled with rapid mixing techniques. For a more detailed structural description of transient folding intermediates, we rely on pulsed hydrogen exchange methods. Finally, we often combine these structural and kinetic methods with mutagenic approaches to investigate the role of individual residues and their interactions in stabilizing the various conformational states that participate in folding.

Another area of interest concerns macromolecular interactions, especially those involved in the recognition of ligand proteins and peptides by antibodies and other proteins. This work again makes extensive use of hydrogen exchange and NMR techniques. Other studies aimed at understanding protein function and molecular recognition on a detailed structural level also rely on modern multidimensional NMR methods.

THE KINETIC IMPORTANCE OF EARLY STRUCTURAL INTERMEDIATES IN PROTEIN FOLDING. RODER, COLÓN, KHORASANIZADEH, PARK, SAUDER, WALKENHORST

Many small proteins can fold into their unique native structures with remarkable speed and efficiency. It has long been realized that the conformational space accessible to an unfolded polypeptide chain is much too vast for a protein to find the native state within such a short time by a random search mechanism. Thus, the prevalent opinion for many years has been that partially folded intermediates are formed at an early stage, limiting the conformational search by directing the polypeptide chain along a productive route toward the native state.

The kinetic importance of folding intermediates has recently been challenged by the observation that some small, single-domain proteins can fold rapidly in an apparent two-state reaction without evidence of structural intermediates. Moreover, there have been theoretical and experimental indications that proteins can encounter misfolded states with non-native conformation. It is unlikely that these observations are general, since there is little evidence for conformationally misfolded states in simple globular proteins. Partially folded kinetic intermediates have, in fact, been observed for many proteins, including those studied by our group. Early intermediates that have been characterized by NMR and hydrogen exchange methods generally show predominantly native-like structural elements, indicating that they are productive intermediates along a direct path toward the native state (1).

Our folding studies on a set of model proteins with diverse structural properties, including mammalian and bacterial cytochromes c, ubiquitin, and staphylococcal nuclease are beginning to reveal some common features. In each case, we find clear evidence for the rapid formation (within a few milliseconds) of folding intermediates with some common structural properties: they are compact states with extensive secondary structure and some critical native-like tertiary contacts, but they lack stable hydrogen bonds and have a loosely packed hydrophobic core. These properties resemble those of the partially folded equilibrium forms (molten globules) found for many proteins under mildly denaturing conditions. For all of the proteins studied in our laboratory, the kinetics of folding and unfolding is consistent with a kinetic mechanism with at least one sequential intermediate that is in rapid equilibrium with unfolded molecules and precedes the rate-limiting step in forming the native structure. Our observations support the notion that condensation of the polypeptide chain at an early stage of folding into a compact, yet dynamic, state with some key attributes of the native structure is essential for rapid and efficient protein folding.

KINETIC ANALYSIS OF UBIQUITIN VARIANTS WITH ALTERED CORE RESIDUES. KHORA-SANIZADEH, RODER, in collaboration with PETERS^e

Ubiquitin, a 76-residue protein with a mixed -sheet and an -helix (Figure 1), is ideal for investigating the intrinsic structural determinants of protein folding.

FIGURE 1. Ribbon diagram of the F45W variant of ubiquitin (4) illustrating the hydrophobic core surrounded by the -helix (residues 23-34) and the mixed 5-strand -sheet. The side chains of Val26 and nearby core resides are shown in ball-and-stick representation. Also shown is the engineered Trp45 side chain, which serves as a fluorescence probe.

U I N (Scheme 1),

where U represents the ensemble of unfolded conformations, the intermediate I represents an ensemble of compact conformations with native-like fluorescence properties, and N is the native state. The intermediate I accumulates under certain stable refolding conditions (low denaturant concentration at room temperature, but not at 8° C) and is in rapid equilibrium with U. Kinetic analysis shows that a denaturant-independent process that limits the rate of folding at low denaturant concentrations reflects the rate-determining conversion of the intermediate into the native state. In terms of its response to mutations and solvent additives, the intermediate resembles the transition state for folding between I and N. The steep decrease in the overall rate of folding observed with increasing denaturant concentration for all ubiquitin variants, as well as most other proteins studied, is attributed to the diminishing population of the intermediate as its structure is destabilized by addition of denaturant.

In contrast to the stable variants, V26L and V26I, the V26A and V26G mutations destabilize not only the native state (by 3.3 and 6.3 kcal/mol, respectively), but also the kinetic intermediate (by 2.2 and 4.3 kcal/mol, respectively). Thus, parts of the hydrophobic core, including the regions around residue 26 and the fluorescence probe Trp45, are already consolidated in the intermediate and the subsequent transition state, although not as tightly packed as in the native structure. The kinetic behavior of V26A and V26G is that of an apparent two-state reaction, even in the absence of denaturant. However, the intermediate reappears for V26A on addition of a stabilizing salt (sodium sulfate), demonstrating that the mutation does not prevent formation of the intermediate, but simply destabilizes it. All observations can be modeled quantitatively with a sequential three-state mechanism (Scheme 1). For the stable variants at low denaturant concentrations, the free energy of I lies between U and N, so that I accumulates as a kinetic intermediate (Figure 2). On the other hand, disruptive mutations and/or addition of denaturants raise the free energy of I above that of U, so that it no longer accumulates as a detectable intermediate, resulting in an apparent two-state folding process with slower rates.

FIGURE 2. Free energy diagrams derived from three-state analysis of the kinetics of ubiquitin folding and unfolding (2), illustrating the effects of solvent conditions (Panel A) and mutation of Val26 to Ala (Panel B) on the relative free energies of the three conformational states, U, I and N.

We also considered an alternative folding mechanism in which I is a non-productive intermediate off the direct path between U and N:

I U N (Scheme 2).

Although formal kinetic analysis cannot distinguish between these alternative models, the sequential mechanism (Scheme 1) requires fewer assumptions to explain the data and is physically more plausible. For example, Scheme 2 predicts that the overall rate of folding should increase when I is destabilized, in contrast to the behavior observed for ubiquitin and other proteins. To fit the data with Scheme 2 requires a large compensating increase in the free energy of the transition state between U and N, while Scheme 1 provides a more straightforward explanation of the observed kinetics in terms of the population of a structural intermediate. Thus, our analysis argues in favor of a protein folding mechanism with productive intermediates, which serve an essential role at early stages of folding in directing the protein toward the native conformation. The model also predicts that proteins showing apparent two-state kinetics may exhibit well-populated kinetic intermediates under sufficiently stabilizing conditions, as we found for the V26A variant of ubiquitin in the presence of a stabilizing salt.

COMPARISON OF THE FOLDING MECHANISMS FOR EUKARYOTIC AND PROKARYOTIC CYTOCHROMES C. SAUDER, RODER, in collaboration with MacKENZIE^f

Aside from a handful of functionally important residues, including a Cys-X-X-Cys-His motif near the N-terminus and a methionine toward the C-terminus involved in heme attachment and ligation, the prokaryotic cytochrome c₂ from Rhodobacter capsulatus has little sequence homology with the mitochondrial cytochromes c. Nevertheless, they exhibit some striking structural similarities, including two -helices near opposite ends of the polypeptide chain that are tightly packed against each other in a perpendicular arrangement. Since formation and association of the N- and C-terminal helices is a critical early event in the folding of horse cytochrome c, it is possible that constraints imposed by the mechanism of folding are responsible for the conservation of this structural motif. Another attractive feature of cytochrome c₂ is that the axial heme ligands, His17 and Met96, are the only histidine and methionine residues in the protein, unlike horse cytochrome c, which has additional histidines and methionines that complicate the folding process by forming non-native heme ligands. It also features a single tryptophan residue in close proximity to the heme (analogous to horse cytochrome c) that serves as a convenient fluorescence probe for monitoring the folding transition.

The kinetics of folding and unfolding of cytochrome c₂ was measured as a function of denaturant concentration, both in the presence and absence of sodium sulfate, which is known to stabilize compact protein conformations (cf. ref. 2). The results are consistent with the following four-state mechanism:

U* I* N* N^M (Scheme 3),

where the asterisk indicates states with only one axial ligand (His17) and a vacant sixth iron coordination site, and N^M represents the native state with two axial ligands (His17 and Met96). I* is a compact intermediate with quenched tryptophan fluorescence, and N* is an almost completely folded state lacking the native Met96-iron bond. I* is well-populated at low denaturant concentrations, where it gives rise to a major burst phase (fluorescence decrease during the mixing dead time), and the folding rate approaches a denaturant-independent value. As in the case of ubiquitin (2), this kinetic behavior is explained by a sequential intermediate in rapid equilibrium with U*, followed by a denaturant-independent rate-limiting step (I* --> N*). The U* I* equilibrium depends strongly on solvent conditions: addition of denaturant shifts it toward U*, so that I* is no longer populated in the unfolding transition region, while addition of sodium sulfate shifts it toward the right, stabilizing I*. In order to explain the unusual denaturant-dependence of the rate of unfolding observed for this and other cytochromes c, it is necessary to introduce a second intermediate, N*, which is encountered in the unfolding process. Above the midpoint of the guanidine-induced unfolding transition, the unfolding reaction is limited by the N* -->; I* step, whereas the N^M --> N* step becomes rate-limiting at very high denaturant concentration. As in the case of horse cytochrome c (3), we attribute this effect to the dissociation of the methionine ligand from the heme, which precedes the main structural unfolding transition.

THE KINETIC ROLE OF HELIX-HELIX PACKING INTERACTIONS IN THE FOLDING OF CYTOCHROME C. COLÓN, ELÖVE, RODER, in collaboration with SHERMAN^g

In order to understand the structural and energetic basis of packing interactions between the N- and C-terminal -helices of horse cytochrome c and their role in folding, we characterized a series of variants in which an important contact residue, Leu94, was replaced by Ile, Val or Ala (3). Last year, we reported preliminary results on the thermodynamic and kinetic consequences of these amino acid changes. We found that the transient population of a previously characterized folding intermediate, I_NC, was especially sensitive to mutations at the helix-helix interface, confirming that it contains tightly interacting N- and C-terminal helices. We have extended these studies by measuring the effect of mutations on the population of an early compact folding intermediate, I_C, which precedes I_NC. Both the population of I_C and the stability of the native structure are quite insensitive to conservative substitutions at position 94 (L94I and L94V), while the I_NC intermediate is strongly destabilized by these mutations. Replacement of Leu94 with the smaller Ala destabilizes all three states and reduces the population of I_NC to negligible levels. These observations show that the initial compaction of the polypeptide chain is driven by relatively nonspecific hydrophobic interactions, while the stabilization of the subsequent I_NC intermediate relies on more specific packing interactions of hydrophobic side chains at the helix-helix interface.

1. RODER, H. Watching protein folding unfold. Nature Struct. Biol. 2: 817-820, 1995.

2. KHORASANIZADEH, S., PETERS, I.D., RODER, H. Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues. Nature Struct. Biol.: 3: 193-205, 1996.

3. COLÓN, W., ELÖVE, G.A., WAKEM, L.P., SHERMAN, F., RODER, H. Side chain packing of the N- and C-terminal helices plays a critical role in the kinetics of cytochrome c folding. Biochemistry 35: 5538-5549, 1996.

   Papers in press at time of previous report:

4. LAUB, P.B., KHORASANIZADEH, S., RODER, H. Localized solution structure refinement of an F45W variant of ubiquitin using stochastic boundary molecular dynamics and NMR distance restraints. Protein Sci. 4: 973-982, 1995.

   ZHANG, Y.Z., PATERSON,Y., RODER, H. Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and 2D NMR. Protein Sci. 4: 804-814, 1995.

^b A.K. Bhuyan: National Center for Biological Sciences, TIFR, Bangalore, 560012, India

^c S. Khorasanizadeh: Chemistry Department, University of Maryland, Baltimore, MD 21228

^d P.B. Laub: Department of Medical Oncology, Fox Chase Cancer Center

^e I.D. Peters: Hawaii Biotechnology Group, Inc., Aiea, HI 96701

^f N.E. MacKenzie: College of Pharmacy, University of Arizona, Tucson, AZ 85721

^g F. Sherman: University of Rochester School of Medicine and Dentistry, Rochester, NY 14642