Mechanism of Thermostabilization in a Designed Cold Shock Protein with Optimized Surface Electrostatic Interactions

Makhatadze, George I.; Loladze, Vakhtang V.; Gribenko, Alexey V.; López, Marı́a M.

doi:10.1016/j.jmb.2003.12.058

Cited by 76 publications

(81 citation statements)

References 82 publications

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“…This spectrum is somewhat unusual, however similar spectra have been observed for CspBs from Bacillus subtilis, 23 Thermotoga maritime, 24 and for major Csp from E. coli, CspA. [25][26][27] Generally, maximum positive band at 200 nm as well as minimum negative band at 225 nm are the characteristics of antiparallel β-sheet structure. Csp exhibits an unusual far UV-CD spectrum for a predominantly β-sheet protein.…”

supporting

confidence: 69%

Recombinant Expression, Isotope Labeling, and Purification of Cold shock Protein from Colwellia psychrerythraea for NMR Study

Moon¹,

Jeong²,

Kim³

et al. 2009

Bulletin of the Korean Chemical Society

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Cold shock proteins (Csps) are a subgroup of the cold-induced proteins on reduction of the growth temperature below the physiological temperature. They preferentially bind to single-stranded nucleic acids to translational regulation via RNA chaperoning. Csp plays important role in cold adaptations for the psychrophilic microorganism. Recently, Cold shock protein from psychrophilic bacteria, Colwellia psychrerythraea (CpCsp) has been identified. Three dimensional structures of a number of Csps from various microorganisms have been solved by NMR spectroscopy or X-ray crystallography, but structures of psychrophilic Csps were not studied yet. Therefore, cloning and purification protocols for further structural study of psychrophilic Csp have been optimized in this study. CpCsp was expressed in E. coli with pET-11a vector system and purified by ion exchange, size exclusion, and reverse phase chromatography. Expression and purification of CpCsp in M9 minimal media was carried out and 15 N-labeled proteins with high purity over 90% was obtained. Further study will be carried out to investigate the tertiary structure and dynamics of CpCsp.

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supporting

confidence: 69%

Recombinant Expression, Isotope Labeling, and Purification of Cold shock Protein from Colwellia psychrerythraea for NMR Study

Moon¹,

Jeong²,

Kim³

et al. 2009

Bulletin of the Korean Chemical Society

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“…Briefly, the goal of computational design was to optimize the overall energy of charge-charge interactions on the protein surface (22). The optimization does not explicitly include salt bridges, which appear to be of a lesser importance (14,18,23,28) than the long-range chargecharge interactions (Figs. S2-S4).…”

Section: Resultsmentioning

confidence: 99%

Modulation of folding energy landscape by charge–charge interactions: Linking experiments with computational modeling

Tzul

Schweiker

Makhatadze

2015

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

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The kinetics of folding-unfolding of a structurally diverse set of four proteins optimized for thermodynamic stability by rational redesign of surface charge-charge interactions is characterized experimentally. The folding rates are faster for designed variants compared with their wild-type proteins, whereas the unfolding rates are largely unaffected. A simple structure-based computational model, which incorporates the Debye-Hückel formalism for the electrostatics, was used and found to qualitatively recapitulate the experimental results. Analysis of the energy landscapes of the designed versus wild-type proteins indicates the differences in refolding rates may be correlated with the degree of frustration of their respective energy landscapes. Our simulations indicate that naturally occurring wild-type proteins have frustrated folding landscapes due to the surface electrostatics. Optimization of the surface electrostatics seems to remove some of that frustration, leading to enhanced formation of native-like contacts in the transition-state ensembles (TSE) and providing a less frustrated energy landscape between the unfolded and TS ensembles. Macroscopically, this results in faster folding rates. Furthermore, analyses of pairwise distances and radii of gyration suggest that the less frustrated energy landscapes for optimized variants are a result of more compact unfolded and TS ensembles. These findings from our modeling demonstrates that this simple model may be used to: (i) gain a detailed understanding of charge-charge interactions and their effects on modulating the energy landscape of protein folding and (ii) qualitatively predict the kinetic behavior of protein surface electrostatic interactions.protein folding | protein stability | charge-charge interaction | energy landscape | computational design T he energy landscape theory provides a conceptual framework to describe the ensemble nature of the protein folding process (1-3). However, a more detailed understanding of contributions from specific types of interactions remains an active area of research (4, 5). Particularly, the question of how interactions between charged residues modulate the funneled energy landscape is not well explored. These interactions are long-range and thus can alter the conformational ensemble at every step of the folding process. The interactions between charged residues are also nonspecific and either attractive or repulsive and therefore their potential effects on the folding energy landscape can be highly complex (6, 7). Traditionally, the modulation of electrostatic interactions in proteins was done by changing the pH or to a lesser degree changing the ionic strength of the solution (8, 9). Such approaches are complicated by the difficulties of predicting the titration properties of individual amino acid residues in the context of ensembles of protein conformations that are sampled during the folding reaction (10). A more attractive approach is to modulate electrostatic interactions via substitutions that perturb the thermody...

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“…In this paper, we present the results of rational design of enzymes with enhanced stability and unchanged enzymatic activity. This approach has 2 major differences from previously described successful protein design methods (2)(3)(4)(5): (i) it concentrates only on the residues on the protein surface, and (ii) it optimizes just one type of interactions, namely, charge-charge interactions on the protein surface (6)(7)(8)(9)(10)(11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

Gribenko

Patel

Liu

et al. 2009

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

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177

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Here, we report the application of a computational approach that allows the rational design of enzymes with enhanced thermostability while retaining full enzymatic activity. The approach is based on the optimization of the energy of charge-charge interactions on the protein surface. We experimentally tested the validity of the approach on 2 human enzymes, acylphosphatase (AcPh) and Cdc42 GTPase, that differ in size (98 vs. 198-aa residues, respectively) and tertiary structure. We show that the designed proteins are significantly more stable than the corresponding WT proteins. The increase in stability is not accompanied by significant changes in structure, oligomerization state, or, most importantly, activity of the designed AcPh or Cdc42. This success of the design methodology suggests that it can be universally applied to other enzymes, on its own or in combination with the other strategies based on redesign of the interactions in the protein core.Until man duplicates a blade of grass, nature will laugh at his so-called scientific knowledge.Thomas Edison computational design ͉ protein engineering ͉ protein stability R ational engineering of proteins to enhance stability and yet retain their enzymatic activity is well motivated (1). One motivation is the practical significance of expanding the use of enzymes in many areas of the modern world, including protein therapeutics, enzymes for food industry, diagnostics, and other areas of industrial biotechnology. Another motivation is validation of the existing scientific knowledge. In this case, predictions made by the existing models for protein stability are subjected to thorough experiments, testing their applicability to protein design. In this paper, we present the results of rational design of enzymes with enhanced stability and unchanged enzymatic activity. This approach has 2 major differences from previously described successful protein design methods (2-5): (i) it concentrates only on the residues on the protein surface, and (ii) it optimizes just one type of interactions, namely, charge-charge interactions on the protein surface (6-15).One of the most important aspects of engineering proteins with enhanced stability, retaining the enzymatic activity, is often forgotten. However, for all of these design efforts to be practically useful, it is important that the engineered proteins retain their biological and enzymatic activity. This issue is particularly important when enhanced protein stability is achieved by redesigning the charge-charge interactions on the protein surface. Such redesign can lead to several potentially detrimental effects on the activity: (i) it can affect the electrostatic potential in the active center, thus reducing or even abolishing the activity; (ii) it can affect substrate/product binding and again reduce or abolish the enzymatic activity; or (iii) it can have effects on the kinetics of substrate binding and, thus, lower the activity via reduced rates of electrostatic steering (3,(16)(17)(18)(19)(20)(21)(22).To this end, it is imp...

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Mechanism of Thermostabilization in a Designed Cold Shock Protein with Optimized Surface Electrostatic Interactions

Cited by 76 publications

References 82 publications

Recombinant Expression, Isotope Labeling, and Purification of Cold shock Protein from Colwellia psychrerythraea for NMR Study

Recombinant Expression, Isotope Labeling, and Purification of Cold shock Protein from Colwellia psychrerythraea for NMR Study

Modulation of folding energy landscape by charge–charge interactions: Linking experiments with computational modeling

Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

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