Robustness of Downhill Folding:  Guidelines for the Analysis of Equilibrium Folding Experiments on Small Proteins

Naganathan, Athi N.; Pérez-Jiménez, Raúl; Sanchez-Ruiz, José M.; Muñoz, Víctor

doi:10.1021/bi050118y

Cited by 81 publications

(171 citation statements)

References 52 publications

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“…Figure 5 shows DSC thermograms for free energy surfaces with a high barrier (~ 18 kJ/mol at the midpoint, see inset), a marginal barrier (~3 kJ/mol at the midpoint, see inset), and fully downhill. The three DSC thermograms can be well fit to a chemical two-state model (continuous lines), as has been discussed before for experimental DSC data (62,65). However, comparison with the true theoretical DSC baseline reveals that the fitted "native" baseline becomes increasingly sloped and shifts to higher values (i.e.…”

Section: Manifestations Of Folding With Marginal (Or No) Barriersmentioning

confidence: 63%

“…A typical example is the interpretation of two observed kinetic phases as an indication of a kinetically populated intermediate. Other deviations are just ignored, or blamed on experimental uncertainties (65). In principle, the marginal barrier and two-state folding regimes could be simply distinguished by measuring the free energy barrier in a DSC experiment.…”

Section: Manifestations Of Folding With Marginal (Or No) Barriersmentioning

confidence: 99%

“…urea). The plot should be linear for a two-state system, while a protein with marginal or no barrier produces a curved profile (65). Another interesting criterion of two-state versus marginal (or no) barrier is provided by the sensitivity to chemical denaturants.…”

Section: Manifestations Of Folding With Marginal (Or No) Barriersmentioning

confidence: 99%

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Dynamics, Energetics, and Structure in Protein Folding

et al. 2006

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For many decades, protein folding experimentalists have worked with no information about the timescales of relevant protein folding motions and without methods for estimating the height of folding barriers. Experiments in protein folding 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 experiment 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 (~14 RT for the very slow folder AcP) to nonexisting. While slow-folding (i.e. 1 millisecond or longer) 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 marginal (or no) barrier can be mapped using equilibrium experiments, which could resolve energetic 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.In folding to their biologically active 3D structures, proteins must coordinate the vast number of degrees of freedom of their polypeptide chains by forming complex networks of noncovalent interactions. Therefore, understanding protein folding involves determining the relations between the energetics of weak interactions and protein conformation, and the collective chain dynamics that govern the search in conformational space. In modern rate theory, these issues are resolved by mapping the potential energy of the molecule as a function of the relevant coordinates. The dynamics are then represented as diffusion on such an energy surface(1,2). For folding reactions, however, even the solvent-averaged free energy surface is hyper-dimensional due to the large number of relevant coordinates (i.e. thousands of atomic coordinates for a small protein)(3). Folding hypersurfaces should also be topographically complex due to frustration between the myriads of possible interactions (3,4). Moreover, molecular simulations(5-8) and NMR dynamics experiments(9) indicate that protein conformational motions span a wide range of timescales (i.e. from picoseconds to milliseconds). The implication is that measuri...

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Section: Manifestations Of Folding With Marginal (Or No) Barriersmentioning

confidence: 63%

Section: Manifestations Of Folding With Marginal (Or No) Barriersmentioning

confidence: 99%

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Dynamics, Energetics, and Structure in Protein Folding

et al. 2006

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“…Those probes that switch signal at lower free energy yield lower melting temperatures. 11,[21][22][23][24][25] In the kinetic approach, the temperature is tuned to identify the appearance of probe-dependent rates and the appearance/ disappearance of nonexponential kinetics. 10,12,26,27 Generally, folding barriers ⌬G † decrease as we tune from the melting temperature T m to the temperature of maximum stability.…”

Section: Introductionmentioning

confidence: 99%

A one-dimensional free energy surface does not account for two-probe folding kinetics of protein α3D

Liu

Dumont

Zhu

et al. 2009

The Journal of Chemical Physics

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We present fluorescence-detected measurements of the temperature-jump relaxation kinetics of the designed three-helix bundle protein ␣ 3 D taken under solvent conditions identical to previous infrared-detected kinetics. The fluorescence-detected rate is similar to the IR-detected rate only at the lowest temperature where we could measure it ͑326 K͒. The fluorescence-detected rate decreases by a factor of 3 over the 326-344 K temperature range, whereas the IR-detected rate remains nearly constant over the same range. To investigate this probe dependence, we tested an extensive set of physically reasonable one-dimensional ͑1D͒ free energy surfaces by Langevin dynamics simulation. The simulations included coordinate-and temperature-dependent roughness, diffusion coefficients, and IR/fluorescence spectroscopic signatures. None of these can reproduce the IR and fluorescence data simultaneously, forcing us to the conclusion that a 1D free energy surface cannot accurately describe the folding of ␣ 3 D. This supports the hypothesis that ␣ 3 D has a multidimensional free energy surface conducive to downhill folding at 326 K, and that it is already an incipient downhill folder with probe-dependent kinetics near its melting point.

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“…In contrast, Fersht and coworkers (15,16) examined the structural, denaturation, and kinetic properties of PSBD homologues from E. coli [unlabeled wild-type BBL, Protein Data Bank (PDB) entry 1W4H], Bacillus stearothermophilus (E3BD F166W, PDB entry 1W4E), and Pyrobaculumaerophilum (POB Y166W L146A, PDB entry 1W4J) (17) and found that 1W4H, 1W4E, and 1W4J follow the typical two-state folding scheme. Whereas Naganathan et al (18) argued that the experimental data in ref. 15 support the global downhill folding scenario, Fersht and coworkers (16) believe that the conclusion drawn by Muñoz and coworkers was obtained by an incorrect assumption and analysis of the data in refs.…”

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confidence: 97%

Cooperative folding kinetics of BBL protein and peripheral subunit-binding domain homologues

Chung

Cheon

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Recent experiments claiming that Naf-BBL protein follows a global downhill folding raised an important controversy as to the folding mechanism of fast-folding proteins. Under the global downhill folding scenario, not only do proteins undergo a gradual folding, but folding events along the continuous folding pathway also could be mapped out from the equilibrium denaturation experiment. Based on the exact calculation using a free energy landscape, relaxation eigenmodes from a master equation, and Monte Carlo simulation of an extended Muñoz-Eaton model that incorporates multiscale-heterogeneous pairwise interactions between amino acids, here we show that the very nature of a two-state cooperative transition such as a bimodal distribution from an exact free energy landscape and biphasic relaxation kinetics manifest in the thermodynamics and folding-unfolding kinetics of BBL and peripheral subunit-binding domain homologues. Our results provide an unequivocal resolution to the fundamental controversy related to the global downhill folding scheme, whose applicability to other proteins should be critically reexamined.protein folding mechanism ͉ global downhill folding ͉ protein thermodynamics ͉ relaxation kinetics of protein O ur understanding of protein folding is primarily based on interpretation of the thermodynamics and kinetics from a simple protein folding model (1-10). The principle of minimal frustration and structural consistency captured by the Go model allowed us to describe the complicated nature of protein folding at a coarse-grained level (5, 6). Bryngelson et al. (6,7) posited a phenomenological picture of free energy landscape of a protein-like heteropolymer and classified protein folding into three scenarios depending on whether the free energy landscape is unistable or bistable and also whether a glass transition occurs. Shakhnovich and Gutin (8) presented microscopic statisticalmechanical theories for heteropolymer model, whose consequences provided important insights into the thermodynamics, kinetics, and nature of free energy landscape for protein folding. The conventional view of protein folding assumes a discrete two-state transition between a native state and a denatured ensemble of protein conformations that are separated by a folding barrier (9). The other view termed as one-state (or global) downhill folding asserts that there is a continuous change of protein conformation during folding without distinct free energy barrier (7, 10). The former could be understood as the first order (cooperative) phase transition whereas the latter as the second order (noncooperative) phase transition between a native and a denatured ensemble of protein conformation. An in-depth analysis for possible downhill folding was performed by Abkevich et al. (10) where it was shown that the major determinant for the cooperativity of protein folding is the relative abundance of nonlocal versus local contacts between residues in the native structure of a protein, which also simultaneously compete with the loss of conform...

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Robustness of Downhill Folding: Guidelines for the Analysis of Equilibrium Folding Experiments on Small Proteins

Cited by 81 publications

References 52 publications

Dynamics, Energetics, and Structure in Protein Folding

Dynamics, Energetics, and Structure in Protein Folding

A one-dimensional free energy surface does not account for two-probe folding kinetics of protein α3D

Cooperative folding kinetics of BBL protein and peripheral subunit-binding domain homologues

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