Victor Muñoz

Spanish National Research Council, Madrid, Madrid, Spain

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Publications (86)603.24 Total impact

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    ABSTRACT: Downhill folding proteins fold in microseconds by crossing a very low or no free energy barrier (<3 RT), and exhibit a complex unfolding behavior in equilibrium. Such unfolding complexity is due to the weak thermodynamic coupling that exists between the various structural segments of these proteins, and it is manifested in unfolding curves that differ depending on the structural probe employed to monitor the process. Probe-dependent unfolding has important practical implications because it permits to investigate the folding energy landscape in detail using multi-probe thermodynamic experiments. This type of thermodynamic behavior has been investigated in depth on the protein BBL, an example of extreme (one-state) downhill folding in which there is no free energy barrier at any condition, including the denaturation midpoint. However, an open question is to what extent is such thermodynamic behavior observed on less extreme downhill folders. Here we perform a multi-probe spectroscopic characterization of the microsecond folder PDD, a structural and functional homolog of BBL that folds within the downhill regime, but is not an example of one-state downhill folding; rather at the denaturation midpoint PDD folds by crossing an incipient free energy barrier. Model-free analysis of the unfolding curves from four different spectroscopic probes together with differential scanning calorimetry reveals a dispersion of ~9 K in the apparent melting temperature and also marked differences in unfolding broadness (from ~50 to ~130 kJ mol-1 when analyzed with a two-state model), confirming that such properties are also observed on less extreme downhill folders. We subsequently perform a global quantitative analysis of the unfolding data of PDD using the same ME statistical mechanical model that was used before for the BBL domain. The analysis shows that this simple model captures all of the features observed on the unfolding of PDD (i.e. the intensity and temperature dependence of the different spectroscopic signals). From the model we estimate a free energy landscape for PDD in which the maximal thermodynamic barrier (i.e. at the denaturation midpoint) is only ~0.5 RT, consistently with previous independent estimates. Our results highlight that multi-probe unfolding experiments in equilibrium combined with statistical mechanical modeling provide important insights into the structural events that take place during the unfolding process of downhill proteins, and thus effectively probe the free energy landscape of these proteins.
    The journal of physical chemistry. B. 07/2014;
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    Fabiana Y Oliva, Victor Muñoz
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    ABSTRACT: The topographic features of the free energy landscapes that govern the thermodynamics and kinetics of conformational transitions in proteins, which in turn are integral for function, are not well understood. This reflects the experimental challenges associated with characterizing these multidimensional surfaces, even for small proteins. Here we focus on a 62-residue protein, gpW, that folds very rapidly into a native structure with an α/β topology in which α-helices are at the N- and C-terminal ends of the molecule with a central β-hairpin positioned orthogonally to the helices. Using relaxation dispersion NMR spectroscopy to probe the conformational fluctuations in gpW at 1 °C, we found that the native state interconverts with a transiently formed, sparsely populated second state with a lifetime of 250 μs, consistent with the global folding-unfolding rate under these conditions. In this low-populated state, the β-hairpin is unfolded whereas the α-helices remain predominantly formed. Our results argue for a hierarchical stability of secondary structural elements and demonstrate the existence of a complex free energy landscape even in this small, fast-folding single-domain protein.
    Journal of the American Chemical Society 05/2014; · 10.68 Impact Factor
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    ABSTRACT: Theory predicts that folding free energy landscapes are intrinsically malleable, and as such are expected to respond to perturbations in topographically complex ways. Structural changes upon perturbation have been observed experimentally for unfolded ensembles, folding transition states, and fast downhill folding proteins. However, the native state of proteins that fold in two-state fashion is conventionally assumed to be structurally invariant during unfolding. Here we investigate how the native and unfolded states of the chicken α-spectrin SH3 domain (a well characterized slow two-state folder) change in response to chemical denaturants and/or temperature. We can resolve the individual properties of the two end-states across the chemical unfolding transition employing single-molecule fluorescence spectroscopy (SM-FRET), and across the thermal unfolding transition by NMR because SH3 folds-unfolds in the slow chemical exchange regime. Our results demonstrate that α-spectrin SH3 unfolds in a canonical way in the sense that it converts between the native state and an unfolded ensemble that expands in response to chemical denaturants. However, as conditions become increasingly destabilizing, the native state also expands gradually; and a large fraction of its native intramolecular hydrogen bonds break up. This gradual disordering of the native state takes place in times shorter than the 100 μs resolution of our SM-FRET experiments. α-spectrin SH3 thus showcases the extreme plasticity of folding landscapes, which extends to the native state of slow two-state proteins. Our results point to the idea that folding mechanisms under physiological conditions might be quite different from those obtained by linear extrapolation from denaturing conditions. Furthermore, they highlight a pressing need for reevaluating the conventional procedures for analyzing and interpreting folding experiments, which may be based on too-simplistic assumptions.
    The Journal of Physical Chemistry B 06/2013; · 3.61 Impact Factor
  • Luis A Campos, Victor Muñoz
    Proceedings of the National Academy of Sciences 04/2013; 110(14):E1242-3. · 9.81 Impact Factor
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    ABSTRACT: A battery of thermodynamic, kinetic, and structural approaches has indicated that the small α-helical protein BBL folds-unfolds via the one-state downhill scenario. Yet, single-molecule fluorescence spectroscopy offers a more conflicting view. Single-molecule experiments at pH 6 show a unique half-unfolded conformational ensemble at mid denaturation, whereas other experiments performed at higher pH show a bimodal distribution, as expected for two-state folding. Here we use thermodynamic and laser T-jump kinetic experiments combined with theoretical modeling to investigate the pH dependence of BBL stability, folding kinetics and mechanism within the pH 6-11 range. We find that BBL unfolding is tightly coupled to the protonation of one of its residues with an apparent pKa of ∼7. Therefore, in chemical denaturation experiments around neutral pH BBL unfolds gradually, and also converts in binary fashion to the protonated species. Moreover, under the single-molecule experimental conditions (denaturant midpoint and 279 K), we observe that proton transfer is much slower than the ∼15 microseconds folding-unfolding kinetics of BBL. The relaxation kinetics is distinctly biphasic, and the overall relaxation time (i.e. 0.2-0.5 ms) becomes controlled by the proton transfer step. We then show that a simple theoretical model of protein folding coupled to proton transfer explains quantitatively all these results as well as the two sets of single-molecule experiments, including their more puzzling features. Interestingly, this analysis suggests that BBL unfolds following a one-state downhill folding mechanism at all conditions. Accordingly, the source of the bimodal distributions observed during denaturation at pH 7-8 is the splitting of the unique conformational ensemble of BBL onto two slowly inter-converting protonation species. Both, the unprotonated and protonated species unfold gradually (one-state downhill), but they exhibit different degree of unfolding at any given condition because the native structure is less stable for the protonated form.
    PLoS ONE 01/2013; 8(10):e78044. · 3.53 Impact Factor
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    ABSTRACT: The study of protein folding has been conventionally hampered by the assumption that all single-domain proteins fold by an all-or-none process (two-state folding) that makes it impossible to resolve folding mechanisms experimentally. Here we describe an experimental method for the thermodynamic analysis of protein folding at atomic resolution using nuclear magnetic resonance (NMR). The method is specifically developed for the study of small proteins that fold autonomously into basic supersecondary structure motifs, and that do so in the sub-millisecond timescale (folding archetypes). From the NMR experiments we obtain hundreds of atomic unfolding curves that are subsequently analyzed leading to the determination of the characteristic network of folding interactions. The application of this approach to a comprehensive catalog of elementary folding archetypes holds the promise of becoming the first experimental approach capable of unraveling the basic rules connecting protein structure and folding mechanism.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 932:205-18. · 1.29 Impact Factor
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    ABSTRACT: Conformational switches are macromolecules that toggle between two states (active/inactive or folded/unfolded) upon specific binding to a target molecule. These molecular devices provide an excellent scaffold for developing real-time biosensors. Here we take this concept one step beyond to build high-performance conformational rheostat sensors. The rationale is to develop sensors with expanded dynamic range and faster response time by coupling a given signal to the continuous (rather than binary) unfolding process of one-state downhill folding protein modules. As proof of concept we investigate the pH and ionic-strength sensing capabilities of the small α-helical protein BBL. Our results reveal that such a pH/ionic-strength sensor exhibits a linear response over 4 orders of magnitude in analyte concentration, compared to the 2 orders of magnitude for switches, and nearly concentration-independent microsecond response times.
    Journal of the American Chemical Society 05/2012; 134(19):8010-3. · 10.68 Impact Factor
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    ABSTRACT: A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 10(6) s(-1)) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out free-diffusion single-molecule FRET experiments with 50-μs resolution and maximal photoprotection using a recently developed Trolox-cysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the one-state downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics.
    Proceedings of the National Academy of Sciences 12/2011; 109(1):179-84. · 9.81 Impact Factor
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    ABSTRACT: The realization that folding free energy barriers can be small enough to result in significant population of the species at the barrier top has sprouted in several methods to estimate folding barriers from equilibrium experiments. Some of these approaches are based on fitting the experimental thermogram measured by differential scanning calorimetry (DSC) to a one-dimensional representation of the folding free-energy surface (FES). Different physical models have been used to represent the FES: (1) a Landau quartic polynomial as a function of the total enthalpy, which acts as an order parameter; (2) the projection onto a structural order parameter (i.e. number of native residues or native contacts) of the free energy of all the conformations generated by Ising-like statistical mechanical models; and (3) mean-field models that define conformational entropy and stabilization energy as functions of a continuous local order parameter. The fundamental question that emerges is how can we obtain robust, model-independent estimates of the thermodynamic folding barrier from the analysis of DSC experiments. Here we address this issue by comparing the performance of various FES models in interpreting the thermogram of a protein with a marginal folding barrier. We chose the small α-helical protein PDD, which folds-unfolds in microseconds crossing a free energy barrier previously estimated as ~1 RT. The fits of the PDD thermogram to the various models and assumptions produce FES with a consistently small free energy barrier separating the folded and unfolded ensembles. However, the fits vary in quality as well as in the estimated barrier. Applying Bayesian probabilistic analysis we rank the fit performance using a statistically rigorous criterion that leads to a global estimate of the folding barrier and its precision, which for PDD is 1.3 ± 0.4 kJ mol(-1). This result confirms that PDD folds over a minor barrier consistent with the downhill folding regime. We have further validated the multi-model Bayesian approach through the analysis of two additional protein systems: gpW, a midsize single-domain with α + β topology that also folds in microseconds and has been previously catalogued as a downhill folder, and α-spectrin SH3, a domain of similar size but with a β-barrel fold, slow-folding kinetics and two-state-like thermodynamics. From a general viewpoint, the Bayesian analysis developed here results in a statistically robust, virtually model-independent, method to estimate the thermodynamic free-energy barriers to protein folding from DSC thermograms. Our method appears to be sufficiently accurate to consistently detect small differences in the barrier height, and thus opens up the possibility of characterizing experimentally the changes in thermodynamic folding barriers induced by single-point mutations on proteins within the downhill regime.
    Physical Chemistry Chemical Physics 07/2011; 13(38):17064-76. · 4.20 Impact Factor
  • David De Sancho, Victor Muñoz
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    ABSTRACT: Protein stability, folding and unfolding rates are all determined by the multidimensional folding free energy surface, which in turn is dictated by factors such as size, structure, and amino-acid sequence. Work over the last 15 years has highlighted the role of size and 3D structure in determining folding rates, resulting in many procedures for their prediction. In contrast, unfolding rates are thought to depend on sequence specifics and be much more difficult to predict. Here we introduce a minimalist physics-based model that computes one-dimensional folding free energy surfaces using the number of aminoacids (N) and the structural class (α-helical, all-β, or α-β) as only protein-specific input. In this model N sets the overall cost in conformational entropy and the net stabilization energy, whereas the structural class defines the partitioning of the stabilization energy between local and non-local interactions. To test its predictive power, we calibrated the model empirically and implemented it into an algorithm for the PREdiction of Folding and Unfolding Rates (PREFUR). We found that PREFUR predicts the absolute folding and unfolding rates of an experimental database of 52 proteins with accuracies of ±0.7 and ±1.4 orders of magnitude, respectively (relative to experimental spans of 6 and 8 orders of magnitude). Such prediction uncertainty for proteins vastly varying in size and structure is only two-fold larger than the differences in folding (±0.34) and unfolding rates (±0.7) caused by single-point mutations. Moreover, PREFUR predicts protein stability with an accuracy of ±6.3 kJ mol(-1), relative to the 5 kJ mol(-1) average perturbation induced by single-point mutations. The remarkable performance of our simplistic model demonstrates that size and structural class are the major determinants of the folding landscapes of natural proteins, whereas sequence variability only provides the final 10-20% tuning. PREFUR is thus a powerful bioinformatic tool for the prediction of folding properties and analysis of experimental data.
    Physical Chemistry Chemical Physics 06/2011; 13(38):17030-43. · 4.20 Impact Factor
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    ABSTRACT: Time resolution of current single-molecule fluorescence techniques is limited to milliseconds because of dye blinking and bleaching. Here we introduce a photoprotection strategy that affords microsecond resolution by combining efficient triplet quenching by oxygen and Trolox with minimized bleaching via the oxygen radical scavenger cysteamine. Using this approach we resolved the single-molecule microsecond conformational fluctuations of two proteins: the two-state folder α-spectrin SH3 domain and the ultrafast downhill folder BBL.
    Nature Methods 02/2011; 8(2):143-6. · 23.57 Impact Factor
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    ABSTRACT: GpW is a 68-residue protein from bacteriophage λ that participates in virus head morphogenesis. Previous NMR studies revealed a novel α+β fold for this protein. Recent experiments have shown that gpW folds in microseconds by crossing a marginal free energy barrier (i.e., downhill folding). These features make gpW a highly desirable target for further experimental and computational folding studies. As a step in that direction, we have re-determined the high-resolution structure of gpW by multidimensional NMR on a construct that eliminates the purification tags and unstructured C-terminal tail present in the prior study. In contrast to the previous work, we have obtained a full manual assignment and calculated the structure using only unambiguous distance restraints. This new structure confirms the α+β topology, but reveals important differences in tertiary packing. Namely, the two α-helices are rotated along their main axis to form a leucine zipper. The β-hairpin is orthogonal to the helical interface rather than parallel, displaying most tertiary contacts through strand 1. There also are differences in secondary structure: longer and less curved helices and a hairpin that now shows the typical right-hand twist. Molecular dynamics simulations starting from both gpW structures, and calculations with CS-Rosetta, all converge to our gpW structure. This confirms that the original structure has strange tertiary packing and strained secondary structure. A comparison of NMR datasets suggests that the problems were mainly caused by incomplete chemical shift assignments, mistakes in NOE assignment and the inclusion of ambiguous distance restraints during the automated procedure used in the original study. The new gpW corrects these problems, providing the appropriate structural reference for future work. Furthermore, our results are a cautionary tale against the inclusion of ambiguous experimental information in the determination of protein structures.
    PLoS ONE 01/2011; 6(11):e26409. · 3.53 Impact Factor
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    ABSTRACT: Proteins fold up by coordinating the different segments of their polypeptide chain through a network of weak cooperative interactions. Such cooperativity results in unfolding curves that are typically sigmoidal. However, we still do not know what factors modulate folding cooperativity or the minimal amount that ensures folding into specific three-dimensional structures. Here, we address these issues on BBL, a small helical protein that folds in microseconds via a marginally cooperative downhill process (Li, P., Oliva, F. Y., Naganathan, A. N., and Muñoz, V. (2009) Proc. Natl. Acad. Sci. USA. 106, 103-108). Particularly, we explore the effects of salt-induced screening of the electrostatic interactions in BBL at neutral pH and in acid-denatured BBL. Our results show that electrostatic screening stabilizes the native state of the neutral and protonated forms, inducing complete refolding of acid-denatured BBL. Furthermore, without net electrostatic interactions, the unfolding process becomes much less cooperative, as judged by the broadness of the equilibrium unfolding curve and the relaxation rate. Our experiments show that the marginally cooperative unfolding of BBL can still be made twice as broad while the protein retains its ability to fold into the native three-dimensional structure in microseconds. This result demonstrates experimentally that efficient folding does not require cooperativity, confirming predictions from theory and computer simulations and challenging the conventional biochemical paradigm. Furthermore, we conclude that electrostatic interactions are an important factor in determining folding cooperativity. Thus, electrostatic modulation by pH-salt and/or mutagenesis of charged residues emerges as an attractive tool for tuning folding cooperativity.
    Journal of Biological Chemistry 11/2010; 285(45):34549-56. · 4.65 Impact Factor
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    ABSTRACT: Proteins that fold over free-energy barriers <or= 3RT are classified as downhill folders. This regime is characterized by equilibrium unfolding that is proportionally broader and more complex the lower the folding barrier. Downhill proteins are also expected to fold up in a few microseconds. However, the relationship between rate and equilibrium signatures is affected by other factors such as protein size and folding topology. Here we perform a direct comparison of the kinetics and equilibrium unfolding of two structural homologues: BBL and PDD. BBL folds-unfolds in just approximately 1 micros at 335 K and displays the equilibrium signatures expected for a protein at the bottom of the downhill folding regime. PDD, which has the same 3D structure and size, folds-unfolds approximately 8 times more slowly and, concomitantly, exhibits all the downhill equilibrium signatures to a lesser degree. Our results demonstrate that the equilibrium signatures of downhill folding are proportional to the changes in folding rate once structural and size-scaling effects are factored out. This conclusion has two important implications: (1) it confirms that the quantitative analysis of equilibrium experiments in ultrafast folding proteins does provide direct information about free-energy barriers, a result that is incompatible with the conventional view of protein folding as a highly activated process, and (2) it advocates for equilibrium-kinetic studies of homologous proteins as a powerful tool to navigate the downhill folding regime via comparative analysis. The latter should prove extremely useful for the investigation of sequence, functional, and evolutionary determinants of protein folding barriers.
    Journal of the American Chemical Society 08/2010; 132(32):11183-90. · 10.68 Impact Factor
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    Athi N Naganathan, Victor Muñoz
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    ABSTRACT: Protein folding mechanisms are probed experimentally using single-point mutant perturbations. The relative effects on the folding (phi-values) and unfolding (1 - phi) 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: DeltaDeltaG(double dagger)(unfold)=0.76DeltaDeltaG(eq) +/- 1.8kJ/mol. Therefore all data are consistent with a single phi-value (0.24) with accuracy comparable to experimental precision, suggesting that the structural information in conventional phi-values is low. (ii) phi-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 phi-values at the mutant denaturation midpoints; i.e., conditions of zero stability (phi(0)). We then show that the intrinsic variability is phi(0) = 0.36 +/- 0.11, being somewhat larger for beta-sheet-rich proteins than for alpha-helical proteins. Importantly, we discover that phi(0)-values are proportional to how many of the residues surrounding the mutated site are local in sequence. High phi(0)-values correspond to protein surface sites, which have few nonlocal neighbors, whereas core residues with many tertiary interactions produce the lowest phi(0)-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 phi-values with many other empirical observations.
    Proceedings of the National Academy of Sciences 04/2010; 107(19):8611-6. · 9.81 Impact Factor
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 30(6).
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    Proceedings of the National Academy of Sciences 12/2009; 106(52):E139; author reply E140. · 9.81 Impact Factor
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    Victor Muñoz
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    ABSTRACT: Sub-microsecond, downhill-reaction protein folding can be investigated by a method to generate large and fast pressure drops. The approach is complementary to nanosecond temperature-jump methods and could provide new insights into the biophysics of protein folding.
    Nature Methods 08/2009; 6(7):490-1. · 23.57 Impact Factor

Publication Stats

4k Citations
603.24 Total Impact Points

Institutions

  • 2009–2013
    • Spanish National Research Council
      • Biological Research Centre
      Madrid, Madrid, Spain
  • 2001–2011
    • University of Maryland, College Park
      • Department of Chemistry and Biochemistry
      College Park, MD, United States
  • 2008
    • University of Bergen
      • Department of Biomedicine
      Bergen, Hordaland Fylke, Norway
    • University of California, San Diego
      San Diego, California, United States
  • 1997–2006
    • National Institutes of Health
      • Laboratory of Chemical Physics (LCP)
      Bethesda, MD, United States
  • 1994–1998
    • European Molecular Biology Laboratory
      • Structural and Computational Biology Unit (Heidelberg)
      Heidelburg, Baden-Württemberg, Germany
    • Instituto de Estructura de la Materia
      Madrid, Madrid, Spain