Victor Muñoz

Elucidating the folding problem of helical peptides using empirical parameters

Using an empirical analysis of experimental data we have estimated a set of energy contributions which accounts for the stability of isolated α-helices. With this database and an algorithm based on statistical mechanics, we describe the average helical behaviour in solution of 323 peptides and the helicity per residue of those peptides analyzed by nuclear magnetic resonance. Moreover the algorithm successfully detects the α-helical tendency, in solution, of a peptide corresponding to a β-strand of ubiquitin.

Atom-by-atom analysis of global downhill protein folding.

Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.

Submillisecond kinetics of protein folding

New experimental methods permit observation of protein folding and unfolding on the previously inaccessible nanosecond-microsecond timescale. These studies are beginning to establish times for the elementary motions in protein folding - secondary structure and loop formation, local hydrophobic collapse, and global collapse to the compact denatured state. They permit an estimate of about one microsecond for the shortest time in which a protein can possibly fold.

How fast is protein hydrophobic collapse

One of the most recurring questions in protein folding refers to the interplay between formation of secondary structure and hydrophobic collapse. In contrast with secondary structure, it is hard to isolate hydrophobic collapse from other folding events. We have directly measured the dynamics of protein hydrophobic collapse in the absence of competing processes. Collapse was triggered with laser-induced temperature jumps in the acid-denatured form of a simple protein and monitored by fluorescence resonance energy transfer between probes placed at the protein ends. The relaxation time for hydrophobic collapse is only ≈60 ns at 305 K, even faster than secondary structure formation. At higher temperatures, as the protein becomes increasingly compact by a stronger hydrophobic force, we observe a slowdown of the dynamics of collapse. This dynamic hydrophobic effect is a high-temperature analogue of the dynamic glass transition predicted by theory. Our results indicate that in physiological conditions many proteins will initiate folding by collapsing to an unstructured globule. Local motions will presumably drive the following search for native structure in the collapsed globule.

Insights into protein folding mechanisms from large scale analysis of mutational effects

Protein folding mechanisms are probed experimentally using single-point mutant perturbations. The relative effects on the folding (ϕ-values) and unfolding (1 - ϕ) rates are used to infer the detailed structure of the transition-state ensemble (TSE). Here we analyze kinetic data on > 800 mutations carried out for 24 proteins with simple kinetic behavior. We find two surprising results: (i) all mutant effects are described by the equation: . Therefore all data are consistent with a single ϕ-value (0.24) with accuracy comparable to experimental precision, suggesting that the structural information in conventional ϕ-values is low. (ii) ϕ-values change with stability, increasing in mean value and spread from native to unfolding conditions, and thus cannot be interpreted without proper normalization. We eliminate stability effects calculating the ϕ-values at the mutant denaturation midpoints; i.e., conditions of zero stability (ϕ0). We then show that the intrinsic variability is ϕ0 = 0.36 ± 0.11, being somewhat larger for β-sheet-rich proteins than for α-helical proteins. Importantly, we discover that ϕ0-values are proportional to how many of the residues surrounding the mutated site are local in sequence. High ϕ0-values correspond to protein surface sites, which have few nonlocal neighbors, whereas core residues with many tertiary interactions produce the lowest ϕ0-values. These results suggest a general mechanism in which the TSE at zero stability is a broad conformational ensemble stabilized by local interactions and without specific tertiary interactions, reconciling ϕ-values with many other empirical observations.

Helix design, prediction and stability

Recent work revealing that our knowledge is now sufficient to build a reasonable quantitative model for the helix/coil transition in heteropolypeptides represents a watershed in research into alpha-helix stability, prediction and design. The opportunity is presented to design specific alpha-helix propensity patterns that may be used both to modify thermodynamic properties of target proteins and peptides, and for de novo protein design. Despite these advances, the picture is not yet complete and further studies of still poorly characterized factors are required to obtain a more precise understanding of alpha-helix stability.

Local versus nonlocal interactions in protein folding and stability – an experimentalist's point of view

One of the classic important issues in protein folding and stability is the relative roles of noncovalent short-range (local) and long-range (nonlocal) interactions. Interest in this topic has been reinforced by recent developments in the analytical theory of protein folding and in lattice-based computer simulations. During the past few years, a wealth of experimental information relevant to this issue has been accumulating. In this review, we focus specifically on experimental aspects, discussing some general ideas that arise from the results obtained by many different groups using a variety of approaches. We also discuss a new experimental strategy that allows us to engineer the contribution of local interactions, and we discuss the first results obtained.

Dynamics of one-state downhill protein folding

The small helical protein BBL has been shown to fold and unfold in the absence of a free energy barrier according to a battery of quantitative criteria in equilibrium experiments, including probe-dependent equilibrium unfolding, complex coupling between denaturing agents, characteristic DSC thermogram, gradual melting of secondary structure, and heterogeneous atom-by-atom unfolding behaviors spanning the entire unfolding process. Here, we present the results of nanosecond T-jump experiments probing backbone structure by IR and end-to-end distance by FRET. The folding dynamics observed with these two probes are both exponential with common relaxation times but have large differences in amplitude following their probe-dependent equilibrium unfolding. The quantitative analysis of amplitude and relaxation time data for both probes shows that BBL folding dynamics are fully consistent with the one-state folding scenario and incompatible with alternative models involving one or several barrier crossing events. At 333 K, the relaxation time for BBL is 1.3 μs, in agreement with previous folding speed limit estimates. However, late folding events at room temperature are an order of magnitude slower (20 μs), indicating a relatively rough underlying energy landscape. Our results in BBL expose the dynamic features of one-state folding and chart the intrinsic time-scales for conformational motions along the folding process. Interestingly, the simple self-averaging folding dynamics of BBL are the exact dynamic properties required in molecular rheostats, thus supporting a biological role for one-state folding.

Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics

Protein folding barriers, which range from zero to the tens of RT that result in classical two-state kinetics, are primarily determined by protein size and structural topology [Plaxco KW, Simons KT, Baker D (1998) J Mol Biol 277:985–994]. Here, we investigate the thermodynamic folding barriers of two relatively large proteins of the same size and topology: bovine α-lactalbumin (BLA) and hen-egg-white lysozyme (HEWL). From the analysis of differential scanning calorimetry experiments with the variable-barrier model [Muñoz V, Sanchez-Ruiz JM (2004) Proc Natl Acad Sci USA 101:17646–17651] we obtain a high barrier for HEWL and a marginal folding barrier for BLA. These results demonstrate a remarkable tuning range of at least 30 kJ/mol (i.e., five to six orders of magnitude in population) within a unique protein scaffold. Experimental and theoretical analyses on these proteins indicate that the surprisingly small thermodynamic folding barrier of BLA arises from the stabilization of partially unfolded conformations by electrostatic interactions. Interestingly, there is clear reciprocity between the barrier height and the biological function of the two proteins, suggesting that the marginal barrier of BLA is a product of natural selection. Electrostatic surface interactions thus emerge as a mechanism for the modulation of folding barriers in response to special functional requirements within a given structural fold.

Expanding the realm of ultrafast protein folding: gpW, a midsize natural single-domain with α+β topology that folds downhill

All ultrafast folding proteins known to date are either very small in size (less than 45 residues), have an alpha-helix bundle topology, or have been artificially engineered. In fact, many of them share two or even all three features. Here we show that gpW, a natural 62-residue alpha+beta protein expected to fold slowly in a two-state fashion, folds in microseconds (i.e., from tau = 33 micros at 310 K to tau = 1.7 micros at 355 K). Thermodynamic analyses of gpW reveal probe dependent thermal denaturation, complex coupling between two denaturing agents, and differential scanning calorimetry (DSC) thermogram characteristic of folding over a negligible thermodynamic folding barrier. The free energy surface analysis of gpW folding kinetics also produces a marginal folding barrier of about thermal energy ( RT) at the denaturation midpoint. From these results we conclude that gpW folds in the downhill regime and is close to the global downhill limit. This protein seems to be poised toward downhill folding by a loosely packed hydrophobic core with low aromatic content, large stabilizing contributions from local interactions, and abundance of positive charges on the native surface. These special features, together with a complex functional role in bacteriophage lambda assembly, suggest that gpW has been engineered to fold downhill by natural selection.