Slow Folding of a Helical Protein: Large Barriers, Strong Internal Friction, or a Shallow, Bumpy Landscape?

The rate at which a protein molecule folds is determined by opposing energetic and entropic contributions to the free energy that shape the folding landscape. Delineating the extent to which they impact the diffusional barrier-crossing events, including the magnitude of internal friction and barrier height, has largely been a challenging task. In this work, we extract the underlying thermodynamic and dynamic contributions to the folding rate of an unusually slow-folding helical DNA-binding domain, PurR, which shares the characteristics of ultrafast downhill-folding proteins but nonetheless appears to exhibit an apparent two-state equilibrium. We combine equilibrium spectroscopy, temperature-viscosity-dependent kinetics, statistical mechanical modeling, and coarse-grained simulations to show that the conformational behavior of PurR is highly heterogeneous characterized by a large spread in melting temperatures, marginal thermodynamic barriers, and populated partially structured states. PurR appears to be at the threshold of disorder arising from frustrated electrostatics and weak packing that in turn slows down folding due to a shallow, bumpy landscape and not due to large thermodynamic barriers or strong internal friction. Our work highlights how a strong temperature dependence on the pre-exponential could signal a shallow landscape and not necessarily a slow-folding diffusion coefficient, thus determining the folding timescales of even millisecond folding proteins and hints at possible structural origins for the shallow landscape.This work was supported by the Wellcome Trust/DBT India Alliance Fellowship IA/I/15/1/501837 awarded to A.N.N. The authors acknowledge the FIST facility sponsored by the Department of Science and Technology (DST), India at the Department of Biotechnology, IITM for the instrumentation. The authors thank Dr. Ramesh L. Gardas for providing access to the viscometer and Somenath Pandey for help with viscosity measurements. Financial support to D.D.S. comes from Eusko Jaurlaritza (Basque Government) through Project IT588-13 and from Grants RYC-2016-19590 and PGC2018-099321-B-I00 from the Spanish Ministry of Science and Universities through the Office of Science Research (MINECO/FEDER)

Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations

Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L\u2192G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity ( 4820 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural-functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance

Protein folding kinetics: barrier effects in chemical and thermal denaturation experiments

10 pages, 5 figures.-- PMID: 17419630 [PubMed].-- PMCID: PMC2527040.-- Author manuscript available in PMC: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17419630Printed version published on May 2, 2007.Recent experimental work on fast protein folding brings about an intriguing paradox. Microsecond-folding proteins are supposed to fold near or at the folding speed limit (downhill folding), but yet their folding behavior seems to comply with classical two-state analyses, which imply the crossing of high free energy barriers. However, close inspection of chemical and thermal denaturation kinetic experiments in fast-folding proteins reveals systematic deviations from two-state behavior. Using a simple one-dimensional free energy surface approach we find that such deviations are indeed diagnostic of marginal folding barriers. Furthermore, the quantitative analysis of available fast-kinetic data indicates that many microsecond-folding proteins fold downhill in native conditions. All of these proteins are then promising candidates for an atom-by-atom analysis of protein folding using nuclear magnetic resonance. We also find that the diffusion coefficient for protein folding is strongly temperature dependent, corresponding to an activation energy of ~1 kJ·mol-1 per protein residue. As a consequence, the folding speed limit at room temperature is about an order of magnitude slower than the ~ 1 μs estimates from high-temperature T-jump experiments. Our analysis is quantitatively consistent with the available thermodynamic and kinetic data on slow two-state folding proteins and provides a straightforward explanation for the apparent fast-folding paradox.This research has been supported by NIH grant GM066800-1 and NSF grant MCB-0317294.Peer reviewe

Predictions from an Ising-like Statistical Mechanical Model on the Dynamic and Thermodynamic Effects of Protein Surface Electrostatics

Charged residues on the surface of a protein are known hot-spots for post-translational modification, protein/ligand-binding, and tuning conformational stabilities. Recent experimental evidence points to the fact that surface electrostatics can also modulate thermodynamic barriers and hence folding mechanisms. To probe for this behavior across different proteins, we develop a novel version of the Wako–Saitô–Muñoz–Eaton (WSME) model in which we include an electrostatic potential term in the energy function while simplifying the treatment of solvation free energy. Both of the energy terms are obtained by quantitatively fitting the model to differential scanning calorimetry (DSC) experiments that carry critical information on the protein partition function. We characterize four sets of structural/functional homologues (HEWL/BLA, CspB, engrailed, α-spectrin) either by fitting the experimental data of a single domain in the homologous set and predicting the conformational behavior of the rest with the same set of parameters or by performing semiblind predictions. The model with the added electrostatic term is able to successfully reproduce the order of thermodynamic stabilities and relaxation rates of most of the homologues. In parallel, we predict diverse conformational features including a wide range of thermodynamic barriers (∼9–40 kJ/mol), broad native ensembles in helical proteins, structured unfolded states and intermediates, rugged folding landscapes, and further provide an independent protein-specific estimate of the folding speed limit at 298 K (1/(7–300 μs)). Our results are evidence that protein surface electrostatics can be tailored to not only engineer stabilities but also folding mechanisms and the ruggedness of the underlying landscape

Determining denaturation midpoints in multiprobe equilibrium protein folding experiments

10 pages, 7 figures.-- PMID: 18540681 [PubMed].Multiprobe equilibrium unfolding experiments in the downhill regime (i.e., maximal barrier < 3RT) can resolve the folding process with atomic resolution [Munoz (2002) Int. J. Quantum Chem. 90, 1522-1528]. Such information is extracted from hundreds of heterogeneous atomic equilibrium unfolding curves, which are characterized according to their denaturation midpoint (e.g., Tm for thermal denaturation). Using statistical methods, we analyze Tm accuracy when determined from the extremum of the derivative of the unfolding curve and from two-state fits under different sets of simulated experimental conditions. We develop simple procedures to discriminate between real unfolding heterogeneity at the atomic level and experimental uncertainty in the single Tm of conventional two-state folding. We apply these procedures to the recently published multiprobe NMR experiments of BBL [Sadqi et al. (2006) Nature 442, 317-321] and conclude that for the 122 single transition atomic unfolding curves reported for this protein the mean Tm accuracy is better than 1.8 K for both methods, compared to the 60 K spread in Tm determined experimentally. Importantly, we also find that when the pre- or posttransition baseline is incomplete, the two-state fits systematically drift the estimated Tm value toward the center of the experimental range. Therefore, the reported 60 K Tm spread in BBL is in fact a lower limit. The derivative method is significantly less sensitive to this problem and thus is a better choice for multiprobe experiments with a broad Tm distribution. The results we obtain in this work lay the foundations for the quantitative analysis of future multiprobe unfolding experiments in fast-folding proteins.This work has been supported by NIH Grant RO1-GM066800, NSF Grant MCB-0317294, and Marie Curie Excellence Award MEXT-CT- 2006-042334.Peer reviewe

Dynamics, energetics, and structure in protein folding

10 pages, 7 figures.-- PMID: 16834320 [PubMed].-- PMCID: PMC2546509.-- Author manuscript available in PMC: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16834320Printed version published on Jul 18, 2006.For many decades, protein folding experimentalists have worked with no information about the time scales of relevant protein folding motions and without methods for estimating the height of folding barriers. Protein folding experiments have been interpreted using chemical models in which the folding process is characterized as a series of equilibria between two or more distinct states that interconvert with activated kinetics. Accordingly, the information to be extracted from experiments was circumscribed to apparent equilibrium constants and relative folding rates. Recent developments are changing this situation dramatically. The combination of fast-folding experiments with the development of analytical methods more closely connected to physical theory reveals that folding barriers in native conditions range from minimally high (~14RT for the very slow folder AcP) to nonexistent. While slow-folding (i.e., ≥ 1 ms) single-domain proteins are expected to fold in a two-state fashion, microsecond-folding proteins should exhibit complex behavior arising from crossing marginal or negligible folding barriers. This realization opens a realm of exciting opportunities for experimentalists. The free energy surface of a protein with a marginal (or no) barrier can be mapped using equilibrium experiments, which could resolve energetic factors from structural factors in folding. Kinetic experiments on these proteins provide the unique opportunity to measure folding dynamics directly. Furthermore, the complex distributions of time-dependent folding behaviors expected for these proteins might be accessible to single-molecule measurements. Here, we discuss some of these recent developments in protein folding, emphasizing aspects that can serve as a guide for experimentalists interested in exploiting this new avenue of research.The research described here has been supported by NIH grant GM-066800 and NSF grant MCB-0317294.Peer reviewe

Thermodynamic architecture and conformational plasticity of GPCRs

GPCRs are integral membrane proteins that serve as attractive drug targets. Here, authors delineate the conformational landscapes of 45 GPCRs using a statistical model, highlighting their malleable native ensembles and providing functional insights

A Rapid, Ensemble and Free Energy Based Method for Engineering Protein Stabilities

Engineering the conformational stabilities of proteins through mutations has immense potential in biotechnological applications. It is, however, an inherently challenging problem given the weak noncovalent nature of the stabilizing interactions. In this regard, we present here a robust and fast strategy to engineer protein stabilities through mutations involving charged residues using a structure-based statistical mechanical model that accounts for the ensemble nature of folding. We validate the method by predicting the absolute changes in stability for 138 experimental mutations from 16 different proteins and enzymes with a correlation of 0.65 and importantly with a success rate of 81%. Multiple point mutants are predicted with a higher success rate (90%) that is validated further by comparing meosphile–thermophile protein pairs. In parallel, we devise a methodology to rapidly engineer mutations in silico which we benchmark against experimental mutations of ubiquitin (correlation of 0.95) and check for its feasibility on a larger therapeutic protein DNase I. We expect the method to be of importance as a first and rapid step to screen for protein mutants with specific stability in the biotechnology industry, in the construction of stability maps at the residue level (i.e., hot spots), and as a robust tool to probe for mutations that enhance the stability of protein-based drugs