Rational Modification of Protein Stability by the Mutation of Charged Surface Residues

Spector, Shari; Wang, Minghui; Carp, Stefan A.; Robblee, James P.; Hendsch, Zachary S.; Fairman, Robert; Tidor, Bruce; Raleigh, Daniel P.

doi:10.1021/bi992091m

Cited by 196 publications

(156 citation statements)

References 41 publications

Supporting

Mentioning

149

Contrasting

Order By: Relevance

“…PB calculations have been used successfully to engineer proteins with higher stability (34,35), but to best of our knowledge the same method as here has not been used much for engineering higher affinity for ligand binding. A related Tanford-Kirkwood electrostatic model (36) has been used in design of specificity of coiled-coil recognition, whereas a previous study (37) used continuum calculations to design cytokine variants with pH-dependent loss of affinity by a histidine-switch.…”

Section: Discussionmentioning

confidence: 99%

Electrostatic Interactions of Hsp-organizing Protein Tetratricopeptide Domains with Hsp70 and Hsp90

Kajander

Sachs

Goldman

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Hsp-organizing protein (HOP) binds to the C termini of the chaperones Hsp70 and Hsp90, thus bringing them together so that substrate proteins can be passed from Hsp70 to Hsp90. Because Hsp90 is essential for the correct folding and maturation of many oncogenic proteins, it has become a significant target for anti-cancer drug design. HOP binds to Hsp70 and Hsp90 via two independent tetratricopeptide (TPR) domains, TPR1 and TPR2A, respectively. We have analyzed ligand binding using Poisson-Boltzmann continuum electrostatic calculations, free energy perturbation, molecular dynamics simulations, and site-directed mutagenesis to delineate the contribution of different interactions to the affinity and specificity of the TPR-peptide interactions. We found that continuum electrostatic calculations could be used to guide protein design by removing unfavorable interactions to increase binding affinity, with an 80-fold increase in affinity for TPR2A. Contributions at buried charged residues, however, were better predicted by free energy perturbation calculations. We suggest using a combination of the two approaches for increasing the accuracy of results, with free energy perturbation calculations used only at selected buried residues of the ligand binding pocket. Finally we present the crystal structure of TPR2A in complex with its non-cognate Hsp70 ligand, which provides insight on the origins of specificity in TPR domain-peptide recognition.The Hsp-organizing protein (HOP) 4 plays a key role in in vivo folding by bringing the chaperones Hsp70 (or its constitutively expressed isoform, Hsp70) and Hsp90 together into a complex (1) (see Fig. 1), where specific substrate proteins are passed over from Hsp70 to Hsp90. Partially folded Hsp90 client proteins are transferred from Hsp70 to Hsp90, where the final steps of folding are completed (2, 3). While bound to the Hsp90, the client protein then becomes functional, e.g. hormone receptors can bind their ligands, and then are released. Both chaperones must be brought together by their interaction with HOP to accomplish this (see Fig. 1). The whole Hsp90 functional cycle involves also other co-chaperones, such as p23, other TPR proteins, and ATP binding and hydrolysis, and is fairly complicated and incompletely understood (4). However, it is clear that HOP facilitates the interaction of Hsp70 and Hsp90 and thus is important in providing the link between the chaperones in the early part of Hps90 functional cycle. Hsp90 client proteins include nuclear hormone receptors, p53, and other transcription factors and various kinases, such as Atk and Her2. Many of these are oncogenes, making Hsp90 a significant anti-cancer drug target (5). It is therefore of considerable importance to better understand the chaperone cycle of Hsp90 and in particular its interaction with essential co-chaperones, such as HOP. The interactions of Hsp70 and Hsp90 with HOP also provide an excellent model system in which to study the contribution of electrostatics to protein-peptide interactions, as will bec...

show abstract

Section: Discussionmentioning

confidence: 99%

Electrostatic Interactions of Hsp-organizing Protein Tetratricopeptide Domains with Hsp70 and Hsp90

Kajander

Sachs

Goldman

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Proteins can be engineered to be more stable in vitro with few mutations, by introduction of specific bonds (39,40) or favorable interactions (41)(42)(43). However, it has been shown that in vitro protein stabilization based on local stabilization of a secondary structural element can strengthen not only the final F state but also a small set of discrete partially folded native-like intermediates (41).…”

Section: Discussionmentioning

confidence: 99%

Hydrogen-exchange stability analysis of Bergerac -Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

Viguera

Serrano

2003

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

View full text Add to dashboard Cite

Amide hydrogen͞deuterium exchange rates have been determined for two mutants of ␣-spectrin Src homology 3 domain (WT), containing an elongated stable (SHH) and unstable (SHA) distal loop. SHA, similarly to WT, follows a two-state transition, whereas SHH apparently folds via a three-state mechanism. Native-state amide hydrogen exchange is effective in ascribing energetic readjustments observed in kinetic experiments to species stabilized within the denatured base and distinguishing those from highenergy barrier crossings. Comparison of ⌬G ex and mex parameters for amide protons of these mutants demonstrates the existence of an intermediate and allows the identification of protons protected in this state. The consolidation of a form containing a prefolded long ␤-hairpin induces the switch to a three-state mechanism in an otherwise two-state folder. It can be inferred that the unbalanced high stability of individual elements of secondary structure in a polypeptide could ultimately complicate its folding mechanism.kinetics ͉ ␤-hairpin T he Src homology 3 (SH3) domain from ␣-spectrin is a wellcharacterized model system for protein folding and stability (1). It was shown early that this domain folds by a two-state transition (2) and that its isolated secondary structure elements are quite unstructured (3). We have investigated (4) the consequences of fusing the SH3 domain with a short sequence KITVNGKTYE (BHH) that tends to fold as a stable ␤-hairpin (5). In the SHHBergerac mutant (SHH for short) the two-residue distal ␤-turn (N47-D48) of ␣-spectrin SH3 has been substituted by BHH. SHH is more stable and folds significantly faster than the short WT ␣-spectrin SH3 at moderate urea concentrations, but unlike WT, its folding rate levels off under strong folding conditions (Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org), thus deviating from the simplest two-state folding mechanism. Mutation of two residues in the inserted BHH region of SHH (Ile-2Ј and Val-4Ј to Ala, SHA-Bergerac) destabilizes the protein and switches it back to a two-state folding process (Fig. 6). When fused to WT, the inserted BHH and BHH I2ЈA V4ЈA sequences adopt a fully folded ␤-hairpin structure protruding from the protein (4), but the corresponding isolated peptides are, respectively, Ϸ30% folded (BHH) and fully unfolded (BHH I2ЈA V4ЈA).Understanding the reasons behind rate constant deviations could be important in improving the methods available for in vitro protein stabilization, as well as in revealing limiting steps related to switches from two to higher order folding mechanisms. Deviations in chevron plots do not have an unequivocal interpretation. Rate constants reflect free energy differences from reactants to the highest energy level in the reaction coordinate or transition state ( ‡). The deviations often occurring at lower denaturant concentrations in refolding experiments are usually assigned to changes within the denatured ensemble, i.e., a new energy level or intermediate state (I) is stabilized...

show abstract

“…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.

210

177

View full text Add to dashboard Cite

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...

show abstract

Rational Modification of Protein Stability by the Mutation of Charged Surface Residues

Cited by 196 publications

References 41 publications

Electrostatic Interactions of Hsp-organizing Protein Tetratricopeptide Domains with Hsp70 and Hsp90

Electrostatic Interactions of Hsp-organizing Protein Tetratricopeptide Domains with Hsp70 and Hsp90

Hydrogen-exchange stability analysis of Bergerac -Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

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

Contact Info

Product

Resources

About