Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation

Babu, Charles R.; Hilser, Vincent J.; Wand, A. Joshua

doi:10.1038/nsmb739

Cited by 143 publications

(220 citation statements)

References 21 publications

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“…The potential advantage of an explicit treatment of cold denaturation is that it will allow the analysis of transitions in which heat and cold denatured states are symmetric (i.e., as predicted by the chemical two-state model) and nonsymmetric (i.e., heat and cold denatured states with different properties). These two scenarios are illustrated by recent work on ubiquitin (26,27). In the presence of guanidine, the heat denatured and cold denatured states of ubiquitin populate in a similar temperature range, and the symmetric scenario holds (26).…”

Section: A Critical Assessment Of the Phenomenological Variable-barriermentioning

confidence: 95%

“…In the presence of guanidine, the heat denatured and cold denatured states of ubiquitin populate in a similar temperature range, and the symmetric scenario holds (26). Without denaturants, heat and cold denaturation are separated by Ͼ100 K, and the two processes appear fundamentally different (27). Our phenomenological model can certainly accommodate both situations by using a more complex expression for the free energy functional.…”

Section: A Critical Assessment Of the Phenomenological Variable-barriermentioning

confidence: 97%

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Exploring protein-folding ensembles: A variable-barrier model for the analysis of equilibrium unfolding experiments

Muñoz

Sanchez-Ruiz²

2004

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

105

154

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Recent theoretical and experimental results point to the existence of small barriers to protein folding. These barriers can even be absent altogether, resulting in a continuous folding transition (i.e., downhill folding). With small barriers, the detailed properties of folding ensembles may become accessible to equilibrium experiments. However, further progress is hampered because folding experiments are interpreted with chemical models (e.g., the twostate model), which assume the existence of well defined macrostates separated by arbitrarily high barriers. Here we introduce a phenomenological model based on the classical Landau theory for critical transitions. In this physical model the height of the thermodynamic free energy barrier and the general properties of the folding ensemble are directly obtained from the experimental data. From the analysis of differential scanning calorimetry data alone, our model identifies the presence of a significant (>35 kJ͞mol) barrier for the two-state protein thioredoxin and the absence of a barrier for BBL, a previously characterized downhill folding protein. These results illustrate the potential of our approach for extracting the general features of protein ensembles from equilibrium folding experiments.experimental analysis ͉ free energy barrier ͉ downhill folding ͉ two-state folding ͉ phenomenological model I n contrast to the situation in many fields of modern physics, experimental results in protein folding are seldom directly interpretable by analytical theory or computer simulations. The interpretation typically involves a simple phenomenological model with which experimental results are analyzed, and the outcome of such ad hoc analysis is used to extract conclusions regarding experiments. A paradigm of this principle is the two-state model, in which protein-folding reactions are analyzed in terms of a chemical equilibrium between two independent species, native (N) and unfolded (U):[1]In Eq. 1, species with intermediate degree of structure are ignored, and the transition from one state to the other is of the first order. The use of a two-state model and its obvious generalization (i.e., a series of chemical equilibria between n structurally defined macrostates) is deeply rooted in the tradition of describing chemical transformations of small molecules as reaction schemes. Despite the obvious limitations of comparing protein folding with simple chemical reactions, the two-state protein-folding model enjoys tremendous popularity. One of the reasons is that this model seems to accommodate the folding behavior of a large set of single-domain proteins (1). Two-state folding could also provide proteins with a significant biological advantage by conferring upon them kinetic stability in vivo (see ref. 2 for a recent discussion). However, to make sure that the two-state character of proteins is not a self-fulfilling prophecy (3), it is important to analyze experimental data with a procedure that does not make assumptions about the existence of a free energy barrier. A more ...

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Section: A Critical Assessment Of the Phenomenological Variable-barriermentioning

confidence: 95%

Section: A Critical Assessment Of the Phenomenological Variable-barriermentioning

confidence: 97%

Exploring protein-folding ensembles: A variable-barrier model for the analysis of equilibrium unfolding experiments

Muñoz

Sanchez-Ruiz²

2004

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

105

154

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“…The populations of nonnative conformational states can be enhanced by changing the temperature, pressure, or pH, or by adding alcohol cosolvents. A cold-denaturation study by Wand et al (41,42) shows for reverse micelle-encapsulated ubiquitin that the mixed ␤-sheet is progressively destabilized from the C-terminal side in the temperature range from Ϫ20°to Ϫ30°C. In an NMR study by Cordier and Grzesiek (43), the change in hydrogen bond strength with increasing temperature was measured.…”

Section: Ubiquitin Unfolding Kinetics Under Equilibrium Conditions Rementioning

confidence: 99%

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Schanda

Forge

Brutscher

2007

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

152

151

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Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of ␣-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of ␣-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes.hydrogen/deuterium exchange ͉ molecular kinetics ͉ ␣-lactalbumin ͉ ubiquitin A detailed description of the structural changes occurring during the folding and unfolding of proteins still remains a challenging objective in biophysics. The understanding of the fundamental mechanisms of the folding process will also shed light on the factors leading to protein misfolding responsible for neurodegenerative diseases such as Alzheimer's and Parkinson's diseases (1). The ideal method to study protein folding/unfolding provides structural information at atomic resolution and is sensitive to changes occurring on time scales ranging from microseconds to minutes, a task that no single technique is able to fulfill. NMR spectroscopy is especially well adapted to obtain detailed atomistic information about the mechanisms, kinetics, and energetics of the folding/unfolding process for virtually every nuclear site in the protein. NMR methods are sensitive to molecular dynamics occurring over a wide range of time scales (Fig. 1a). Although steady-state NMR methods are well suited to characterize equilibrium molecular dynamics occurring on a subsecond time scale (2, 3), unidirectional processes are best studied by real-time NMR (4, 5), where a series of NMR spectra is recorded during the reaction (Fig. 1b). Two difficulties have so far hampered the widespread application of real-time NMR to the study of protein folding. The first problem is the low intrinsic sensitivity of NMR at ambient temperature, a consequence of the small transition energies ...

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“…Two kinds of encapsulation are now widely used. The first is formed by nanoporous silica gels or glasses [32][33][34][35] or polyacrylamide gels [36]; the second is formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles [37][38][39][40][41]. In both cases, the cage sizes can be controlled.…”

Section: Experimental Studies In the Test Tube Enhancement Of Foldingmentioning

confidence: 99%

Protein folding in confined and crowded environments

Zhou

2008

Archives of Biochemistry and Biophysics

151

137

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Confinement and crowding are two major factors that can potentially impact protein folding in cellular environments. Theories based on considerations of excluded volumes predict disparate effects on protein folding stability for confinement and crowding: confinement can stabilize proteins by over 10k B T but crowding has a very modest effect on stability. On the other hand, confinement and crowding are both predicted to favor conformations of the unfolded state which are compact, and consequently may increase the folding rate. These predictions are largely borne out by experimental studies of protein folding under confined and crowded conditions in the test tube. Protein folding in cellular environments is further complicated by interactions with surrounding surfaces and other factors. Concerted theoretical modeling and test-tube and in vivo experiments promise to elucidate the complexity of protein folding in cellular environments.The complexity of the cellular environments in which proteins function is increasingly been appreciated [1]. Potential impacts of two related, but distinct factors, confinement and macromolecular crowding, on protein folding are receiving extensive attention [2][3][4][5]. Confinement arises from encapsulation of proteins in compartments with dimensions on the scales of 10's to 100's Å, which are only moderately larger than the sizes of the proteins themselves. Examples of such compartments include the Anfinsen cage of chaperonins and the exit tunnel of ribosomes. Crowding refers to the exclusion of volumes to proteins of interest by the presence of other macromolecules. The aims of this paper are to discuss potential impacts of confinement and crowding on protein folding and to review the progress made by recent studies on protein folding in confined and crowded conditions and in cellular environments.

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Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation

Cited by 143 publications

References 21 publications

Exploring protein-folding ensembles: A variable-barrier model for the analysis of equilibrium unfolding experiments

Exploring protein-folding ensembles: A variable-barrier model for the analysis of equilibrium unfolding experiments

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Protein folding in confined and crowded environments

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