How enzymes adapt: lessons from directed evolution

Arnold, Frances H.; Wintrode, Patrick L.; Miyazaki, Kentaro; Gershenson, Anne

doi:10.1016/s0968-0004(00)01755-2

Cited by 345 publications

(199 citation statements)

References 30 publications

Supporting

Mentioning

191

Contrasting

Unclassified

Order By: Relevance

“…S2A. Likewise, in the absence of selection substitutions are more often destabilizing than stabilizing (binomial test, P < 10 −15 ), as expected from empirical studies on the effects of random mutations (61,72,75,76,80).…”

Section: Resultsmentioning

confidence: 59%

“…Random mutations in a protein-coding sequence typically destabilize the protein structure (26,72,(75)(76)(77)(78)(79)(80). Thus, if protein evolution proceeded solely via random substitutions, without any selection, we would expect a decrease in protein stability over time.…”

Section: Resultsmentioning

confidence: 99%

“…Our model of purifying selection assumes that overstabilizing mutations are as deleterious as destabilizing mutations, so that only the wild-type stability has optimal fitness. However, several studies have shown that overstabilizing mutations can be neutral under stabilizing selection (75,77,80,88). Therefore, we also considered an alternative, semi-Gaussian fitness landscape in which argT sequences more stable than the wild type are just a fit as the wild type (Fig.…”

Section: Robustness Of Simulation Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Contingency and entrenchment in protein evolution under purifying selection

Shah

McCandlish

Plotkin

2015

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

170

226

View full text Add to dashboard Cite

The phenotypic effect of an allele at one genetic site may depend on alleles at other sites, a phenomenon known as epistasis. Epistasis can profoundly influence the process of evolution in populations and shape the patterns of protein divergence across species. Whereas epistasis between adaptive substitutions has been studied extensively, relatively little is known about epistasis under purifying selection. Here we use computational models of thermodynamic stability in a ligand-binding protein to explore the structure of epistasis in simulations of protein sequence evolution. Even though the predicted effects on stability of random mutations are almost completely additive, the mutations that fix under purifying selection are enriched for epistasis. In particular, the mutations that fix are contingent on previous substitutions: Although nearly neutral at their time of fixation, these mutations would be deleterious in the absence of preceding substitutions. Conversely, substitutions under purifying selection are subsequently entrenched by epistasis with later substitutions: They become increasingly deleterious to revert over time. Our results imply that, even under purifying selection, protein sequence evolution is often contingent on history and so it cannot be predicted by the phenotypic effects of mutations assayed in the ancestral background.intragenic epistasis | coevolution | near neutrality | protein stability W hether a heritable mutation is advantageous or deleterious to an organism often depends on the evolutionary history of the population. A mutation that is beneficial at the time of its introduction may confer its beneficial effect only in the presence of other potentiating or permissive mutations (1-9). Thus, the fate of a mutation arising in a population may be contingent on previous mutations (10-13). Conversely, once a mutation has fixed in a population, the mutation becomes part of the genetic background onto which subsequent modifications are introduced. Because the beneficial effects of the subsequent modifications may depend on the focal mutation, as time passes reversion of the focal mutation may become increasingly deleterious, leading to a type of evolutionary conservatism, or entrenchment (14-18).In the context of protein evolution, the effects of contingency and entrenchment are most easily studied by considering a sequence of single amino acid changes (19) that extends both forward and backward in time from some focal substitution. To assess the roles of contingency and entrenchment we can study the degree to which each focal substitution was facilitated by previous substitutions, and the degree to which the focal substitution influences the subsequent course of evolution (Fig. 1A).Dependencies within a sequence of substitutions are closely connected to the concept of epistasis-that is, the idea that the phenotypic effect of a mutation at a particular genetic site may depend on the genetic background in which it arises (20-24). In the absence of epistasis, a mutation has the same effect ...

show abstract

Section: Resultsmentioning

confidence: 59%

Section: Resultsmentioning

confidence: 99%

Section: Robustness Of Simulation Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Contingency and entrenchment in protein evolution under purifying selection

Shah

McCandlish

Plotkin

2015

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

170

226

View full text Add to dashboard Cite

show abstract

“…Moreover, in many cases, WT-residues have the maximal or second maximal P i , and yet the substitutions in these residues lead to increase of protein stability without changes in enzymatic activity. One possible explanation of these observations is that the interactions with other proteins and ligands in the cell dictate the preference for the residues at these positions, i.e., activity-forstability tradeoff (37,38). Although cellular partners interacting with AcPh are not known, proteins interacting with Cdc42 are well characterized, and disruption of these interactions frequently leads to lethal phenotype.…”

Section: Resultsmentioning

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.

205

175

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

“…Furthermore, many researchers have attempted to improve enzyme stability and to enhance enzyme activity by rational design and in vitro evolution of enzymes. [19][20][21][22][23] In most cases, however, protein biocatalysts showing the desired properties have not been obtained.…”

Section: Introductionmentioning

confidence: 99%

Enhancement of enzyme activity and stability by poly(γ-glutamic acid)

Lee

Tsujimoto

Uyama

et al. 2010

Polym J

View full text Add to dashboard Cite

The effects of poly(c-glutamic acid) (cPGA), a water-soluble poly(amino acid), on the enzyme activity and stability have been investigated. The activity of carbonic anhydrase (CA) was distinctly improved in the presence of 0.30-2.0% cPGA at pH 7. cPGA was also effective to enhance the activity of lipase and a-amylase. cPGA efficiently suppressed denaturation of the enzyme by the thermal treatment and repeated freeze-thaw process. The interaction of cPGA and CA was investigated by using fluorescence probe-labeled protein. The Michaelis-Menten kinetics on the CA-catalyzed hydrolysis of p-nitrophenyl acetate in the absence or presence of cPGA were performed, which demonstrated that the enhancement of the CA activity by cPGA is ascribed to the increase of the catalytic constant k cat .

show abstract

How enzymes adapt: lessons from directed evolution

Cited by 345 publications

References 30 publications

Contingency and entrenchment in protein evolution under purifying selection

Contingency and entrenchment in protein evolution under purifying selection

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

Enhancement of enzyme activity and stability by poly(γ-glutamic acid)

Contact Info

Product

Resources

About