Protein Stability and Surface Electrostatics:  A Charged Relationship

Strickler, Samantha; Gribenko, Alexey V.; Gribenko, Alexander V; Keiffer, Timothy R.; Tomlinson, Jessica M.; Reihle, Tracey; Loladze, Vakhtang V.; Makhatadze, George I.

doi:10.1021/bi0600143

Cited by 300 publications

(269 citation statements)

References 43 publications

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“…2 A and C. The favorable energies of charge-charge interactions increase with the number of substitutions until reaching saturation values. Such saturation behavior has been observed for other proteins and is consistent with 2 facts (7,8,29): (i) for a given protein topology, there is a finite number of surface sites where charged residues can be introduced, and (ii) addition of a charged residue at a new site has both favorable (with residues of the opposite charge) and unfavorable (with residues of the same charge) interactions.…”

Section: Resultssupporting

confidence: 82%

“…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).…”

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confidence: 99%

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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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210

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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Section: Resultssupporting

confidence: 82%

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.

Self Cite

210

177

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“…For example the pH dependent stability of the C-terminal domain of L9 can be well accounted for by assuming unperturbed DES pK a values [34]. In addition the ΔΔG 0 values for a range of mutations in a number of proteins have been well predicted by considering only native state electrostatic interactions suggesting that DSE electrostatic interactions may not be critical in these systems [35].…”

Section: Electrostatic Interactions Are Found In the Denatured State mentioning

confidence: 99%

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

Cho¹,

Sato²,

Horng³

et al. 2008

Archives of Biochemistry and Biophysics

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It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol −1 to the stability of the DSE.

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“…3b). Surface charge-charge relationships are also important in maintaining the stability of the protein [Strickler et al, 2006].…”

Section: Mutations A¡ecting the Electrostatic Surface Potential Of Hnementioning

confidence: 99%

Bioinformatic analysis of protein structure–function relationships: case study of leukocyte elastase (ELA2) missense mutations

Thusberg

Vihinen

2006

Hum. Mutat.

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For the Immunogenetics Special IssueCyclic and congenital neutropenia are caused by mutations in the human neutrophil elastase (HNE) gene (ELA2), leading to an immunodeficiency characterized by decreased or oscillating levels of neutrophils in the blood. The HNE mutations presumably cause loss of enzyme activity, consequently leading to compromised immune system function. To understand the structural basis for the disease, we implemented methods from bioinformatics to analyze all the known HNE missense mutations at both the sequence and structural level. Our results demonstrate that the 32 different mutations have diverse effects on HNE structure and function, affecting structural disorder and aggregation tendencies, stability maintaining contacts, and electrostatic properties. A large proportion of the mutations are located at conserved amino acids, which are usually essential in determining protein structure and function. The majority of the disease-causing HNE missense mutations lead to major structural changes and loss of stability in the protein. A few mutations also affect functional residues, leading into decreased catalytic activity or altered ligand binding. Our analysis reveals the putative effects of all known missense mutations in HNE, thus allowing the structural basis of cyclic and congenital neutropenia to be elucidated. We have employed and analyzed a set of some 30 different methods for predicting the effects of amino acid substitutions. We present results and experience from the analysis of the applicability of these methods in the analysis of numerous genes, proteins, and diseases to reveal protein structure-function relationships and disease genotype-phenotype correlations.

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Protein Stability and Surface Electrostatics: A Charged Relationship

Cited by 300 publications

References 43 publications

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

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

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

Bioinformatic analysis of protein structure–function relationships: case study of leukocyte elastase (ELA2) missense mutations

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