Surface point mutations that significantly alter the structure and stability of a protein's denatured state

Smith, Catherine K.; Bu, Zimei; Anderson, Karen S.; Sturtevant, Julian M.; Engelman, Donald M.; Regan, Lynne

doi:10.1002/pro.5560051007

Cited by 56 publications

(48 citation statements)

References 46 publications

(50 reference statements)

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“…Previous work has shown that introducing nonnative local structural propensity into a protein can lead to a compaction of the unfolded state (35). In addition, a single Ala-to-Thr substitution in the ␤-sheet of a variant B1 domain of protein G caused an increase in folding rate and compaction of the unfolded state, as assessed by the m f3u values derived from folding kinetics experiments (36). A point that argues against a significant alteration in the unfolded-state structures of the Gly-48 mutants is that their m f3u values do not deviate from the WT value.…”

Section: Discussionmentioning

confidence: 99%

Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

Nardo

Korzhnev

Stogios

et al. 2004

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

100

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Through a mutagenic investigation of Gly-48, a highly conserved position in the Src homology 3 domain, we have discovered a series of amino acid substitutions that are highly destabilizing, yet dramatically accelerate protein folding, some up to 10-fold compared with the wild-type rate. The unique folding properties of these mutants allowed for accurate measurement of their folding and unfolding rates in water with no denaturant by using an NMR spin relaxation dispersion technique. A strong correlation was found between ␤-sheet propensity and the folding rates of the Gly-48 mutants, even though Gly-48 lies in an unusual non-␤-strand backbone conformation in the native state. This finding indicates that the accelerated folding rates of the Gly-48 mutants are the result of stabilization of a nonnative ␤-strand conformation in the transition-state structure at this position, thus providing the first, to our knowledge, experimentally elucidated example of a mechanism by which folding can occur fastest through a nonnative conformation. We also demonstrate that residues that are most stabilizing in the transition-state structure are most destabilizing in the native state, and also cause the greatest reductions in in vitro functional activity. These data indicate that the unusual native conformation of the Gly-48 position is important for function, and that evolutionary selection for function can result in a domain that folds at a rate far below the maximum possible. T he energetics involved as proteins progress from a broad ensemble of unfolded conformations to a single native structure are still not thoroughly understood. A widely used approach to study folding is the protein engineering method, which measures the thermodynamic and kinetic effects of single amino acid substitutions (usually with Ala) at many different sites in a protein as a means to define the most structured regions of the folding transition state (1, 2). Mapping the transition state identifies regions that fold more quickly than others, and such knowledge has led to important theoretical advances in our understanding of the folding process (3, 4). Although this method provides a general picture of folding pathways, it does not define the mechanisms by which individual residues can facilitate or retard the folding process. Our approach to study these mechanisms is to investigate the effects of multiple amino acid substitutions at single positions (5-7). In the present study, this approach is used to investigate the role in folding and function of position 48 of the Src homology 3 (SH3) domain, which is one of most conserved positions in this domain (8).The SH3 domain is well suited for protein-folding studies due to its small size and amenability to biophysical analysis (9-11). Composed of two three-stranded ␤-sheets packed orthogonally against one another (Fig. 1A), SH3 domains function as proteinprotein interaction modules in a wide variety of eukaryotic proteins. The structure of the SH3 domain transition state has been extensively characterized...

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Section: Discussionmentioning

confidence: 99%

Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

Nardo

Korzhnev

Stogios

et al. 2004

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

100

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“…The values thus calculated may be interpreted [see Smith et al (1996)] as the apparent radius of gyration of the unfolded state (hence the subindex U we have used in eq 12). The results of the calculation (eq 12) for the urea-induced unfolding of HEW lysozyme at pH 2.9 are shown in Figure 8.…”

Section: Experimental Data Reported In This Work Do Not Provide Suppomentioning

confidence: 99%

Are There Equilibrium Intermediate States in the Urea-Induced Unfolding of Hen Egg-White Lysozyme?

Ibarra‐Molero

Sanchez-Ruiz

1997

Biochemistry

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Protein folding intermediates that are sometimes populated at equilibrium under mild denaturing conditions have attracted much attention as plausible models for the kinetic intermediates transiently populated in the refolding kinetic pathways. Hen egg-white lysozyme is often considered as a typical example of close adherence to the equilibrium, two-state unfolding mechanism. However, recent smallangle X-ray scattering studies suggest that an equilibrium intermediate state is significantly populated in the urea-induced unfolding of this protein at moderately acidic pH. In this work, we analyze the ureainduced unfolding of hen egg-white lysozyme on the basis of steady-state fluorescence measurements, characterization of the folding-unfolding kinetics, double-jump unfolding assays for the amount of native protein, and double-jump refolding assays for the amount of unfolded protein. Our results do not provide support for the presence of an intermediate state and, in particular, disfavor that the following two types of intermediates be significantly populated at equilibrium: (1) intermediates showing a substantial quenching of the tryptophan fluorescence (such as that observed in the transient intermediates found in the refolding kinetic pathway under strongly native conditions) and (2) associating intermediates. Also, the deconvolution of the radius of gyration unfolding profile by using the values for the amount of native state derived from our double-jump unfolding assays is consistent with a two-state unfolding equilibrium and suggests, furthermore, that, in this case, large alterations in the average structure of the unfolded ensemble do not take place in response to changes in urea concentration. This work points up possible pitfalls in the experimental detection of equilibrium folding intermediates and suggests procedures to circumvent them.

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“…The occurrence of a collapse preceding protein folding was inferred from kinetic experiments (6)(7)(8)(9)(10)(11) and also reported in equilibrium experiments, using small-angle x-ray scattering (SAXS) (12) and single-molecule fluorescence (13,14). However, the thermodynamics of the CG transition have not been characterized yet.…”

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confidence: 94%

Coil–globule transition in the denatured state of a small protein

Sherman

Haran

2006

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

287

383

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Upon transfer from strongly denaturing to native conditions, proteins undergo a collapse that either precedes folding or occurs simultaneously with it. This collapse is similar to the well known coil-globule transition of polymers. Here we employ singlemolecule fluorescence methods to fully characterize the equilibrium coil-globule transition in the denatured state of the IgGbinding domain of protein L. By using FRET measurements on freely diffusing individual molecules, we determine the radius of gyration of the protein, which shows a gradual expansion as the concentration of the denaturant, guanidinium hydrochloride, is increased all the way up to 7 M. This expansion is observed also in fluorescence correlation spectroscopy measurements of the hydro- fluorescence correlation spectroscopy ͉ protein folding ͉ single-molecule fluorescence T he coil-globule (CG) transition is a hallmark of the physics of polymers in solution. When a polymer molecule is transferred from a good solvent to a bad one, it undergoes a collapse from an expanded coil-like conformation to a contracted, globule-like conformation. This collapse is typically a second-order phase transition and can be accounted well by mean-field theory (1, 2). Proteins are heteropolymers and therefore should exhibit a similar transition. Because proteins also undergo a first-order folding transition to form their native structure, it is of prime interest to deduce the relation between collapse and folding (3). If the CG transition precedes folding significantly, then the final rearrangements of the protein chain to form the native structure occur within a relatively limited configurational space, which might affect the efficiency and speed of the process. The CG transition of proteins is also important for the elucidation of the properties of their denatured states. Chain collapse can have an impact on secondary-structure formation in the denatured protein (4). Determining the energetics of the collapse, which involve changes in the solvation energy of the protein as the solution conditions are varied, might allow us to understand better the role of the denatured state in the folding transition (5).The occurrence of a collapse preceding protein folding was inferred from kinetic experiments (6-11) and also reported in equilibrium experiments, using small-angle x-ray scattering (SAXS) (12) and single-molecule fluorescence (13,14). However, the thermodynamics of the CG transition have not been characterized yet. In this work, we use two methods, singlemolecule FRET (smFRET) and fluorescence correlation spectroscopy (FCS), to measure the CG transition of a 64-amino acid protein exhibiting two-state folding, the IgG-binding domain of protein L (hereafter denoted simply as protein L) (15). The CG transition is driven by the chemical denaturant guanidinium hydrochloride (GuHCl). We then employ a version of the mean-field theory of Sanchez (16) to obtain a full thermodynamic characterization of the CG transition of a protein, identifying the transition point as well ...

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Surface point mutations that significantly alter the structure and stability of a protein's denatured state

Cited by 56 publications

References 46 publications

Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

Are There Equilibrium Intermediate States in the Urea-Induced Unfolding of Hen Egg-White Lysozyme?

Coil–globule transition in the denatured state of a small protein

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