Protein stability, flexibility and function

Teilum, Kaare; Olsen, Johan G.; Kragelund, Birthe B.

doi:10.1016/j.bbapap.2010.11.005

Cited by 187 publications

(132 citation statements)

References 72 publications

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“…1B), so that both destabilizing and overstabilizing mutations produce variants with lower fitness than the wild type. This assumption is consistent with empirical measurements on several families of proteins (66)(67)(68)(69). In addition, we consider an alternative, semi-Gaussian fitness function that penalizes only destabilizing mutations (discussed below).…”

Section: Modelsupporting

confidence: 51%

Contingency and entrenchment in protein evolution under purifying selection

Shah

McCandlish

Plotkin

2015

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

180

236

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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 ...

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Section: Modelsupporting

confidence: 51%

Contingency and entrenchment in protein evolution under purifying selection

Shah

McCandlish

Plotkin

2015

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

180

236

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“…The acquisition of amino acid substitutions due to mutations can change the properties of proteins to provide an altered function and a selective advantage for an organism (38,39). Most proteins, however, are marginally stable entities and can become unstable in the face of amino acid substitutions (7,13,40). Therefore, stability itself imposes limits on the evolution of protein function.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases

Patel

Fryszczyn

Palzkill

2015

Antimicrob Agents Chemother

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The widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of ␤-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active siteassociated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 ␤-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extendedspectrum ␤-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of ␤-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levels in vivo and protein stability in vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in ␤-lactamase evolution and a common mechanism in protein evolution.T he introduction of new antibiotics into clinical use fuels the evolution of enzymes causing drug resistance. These enzymes gain the ability to inactivate antibiotics more efficiently by acquiring point mutations in and around their active sites that increase catalytic activity (1). However, the introduction of new gain-offunction mutations within highly organized active sites often comes at a stability cost to the enzymes (2-6). Therefore, enzymes can absorb only a limited number of destabilizing mutations before they unfold and lose function (7). This limitation in the evolution of catalytic efficiency is often overcome by the acquisition of global stabilizing mutations that offset the incremental loss in stability due to the primary mutation. This compensation mechanism allows the enzymes to continue a mutational trajectory, resulting in increased resistance.The role of global stabilizing mutations in enzyme evolution has been widely studied (4,8,9). In recent decades, the selection of mutant ␤-lactamase enzymes with expanded substrate specificity has occurred for the plasmid-mediated TEM-and SHV-type ␤-lactamases to give rise to extended-spectrum ␤-lactamases (ESBLs) (10, 11). These enzymes arise due to mutations resulting in substitutions near the active site that increase hydrolysis of oxyimino-cephalosporins such as cefotaxime and ceftazidime. The substitutions that increa...

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“…(1,2) These proteins must, within a well determined tridimensional complex structure, subtly balance thermodynamic stability with conformational flexibility. (3,4) Thermodynamic stability is required to maintain a protein's activity throughout its lifetime while preventing proteolysis or aggregation. In parallel, flexibility is necessary for newly synthesized chains to acquire the native conformation, to assemble and afterwards for the protein to perform its function.…”

Section: Introductionmentioning

confidence: 99%

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

2017

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Most oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the WT and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding/folding by Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) at peptide resolution and by additional biochemical techniques. We found that WT and D93N native hexamers present a stable core and a flexible periphery, the latter being more flexible for the destabilized mutant. Stable but inactive species formed during unfolding of D93N and folding of WT were characterized. For the first time, we show that both of these species are native-like dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α 0 ) remains unfolded. The Kpn/α 0 subdomain, which belongs to the catalytic site, becomes structured only upon hexamerization, explaining why oligomerization is required for NDPK activity. Further HDX-MS studies are necessary to establish the general activation mechanism for other homo-oligomers.

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Protein stability, flexibility and function

Cited by 187 publications

References 72 publications

Contingency and entrenchment in protein evolution under purifying selection

Contingency and entrenchment in protein evolution under purifying selection

Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

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