Article critique of Epistasis in protein evolution

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you have to write an Article critique which covers some aspect of Evolution.

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Article critique of Epistasis in protein evolution
Article critique of Epistasis in protein evolution

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REVIEWS Epistasis in protein evolution Tyler N. Starr1 and Joseph W. Thornton2* 1 Graduate Program in Biochemistry and Molecular Biophysics, University of Chicago, Chicago, Illinois 60637 2 Departments of Ecology and Evolution and Human Genetics, University of Chicago, Chicago, Illinois 60637 Received 2 December 2015; Accepted 27 January 2016 DOI: 10.1002/pro.2897 Published online 2 February 2016 Abstract: The structure, function, and evolution of proteins depend on physical and genetic interactions among amino acids. Recent studies have used new strategies to explore the prevalence, biochemical mechanisms, and evolutionary implications of these interactions—called epistasis—within proteins. Here we describe an emerging picture of pervasive epistasis in which the physical and biological effects of mutations change over the course of evolution in a lineage-specific fashion. Epistasis can restrict the trajectories available to an evolving protein or open new paths to sequences and functions that would otherwise have been inaccessible. We describe two broad classes of epistatic interactions, which arise from different physical mechanisms and have different effects on evolutionary processes. Specific epistasis—in which one mutation influences the phenotypic effect of few other mutations—is caused by direct and indirect physical interactions between mutations, which nonadditively change the protein’s physical properties, such as conformation, stability, or affinity for ligands. In contrast, nonspecific epistasis describes mutations that modify the effect of many others; these typically behave additively with respect to the physical properties of a protein but exhibit epistasis because of a nonlinear relationship between the physical properties and their biological effects, such as function or fitness. Both types of interaction are rampant, but specific epistasis has stronger effects on the rate and outcomes of evolution, because it imposes stricter constraints and modulates evolutionary potential more dramatically; it therefore makes evolution more contingent on low-probability historical events and leaves stronger marks on the sequences, structures, and functions of protein families. Keywords: epistasis; evolutionary biochemistry; sequence-function relationship; protein evolution; sequence space; deep mutational scanning; ancestral sequence reconstruction Introduction A protein’s biological functions emerge from its chemical and physical properties, which in turn are determined by the interactions between its amino acid residues in three-dimensional space. It is thereGrant sponsor: NIH; Grant number: R01GM104397; Grant sponsor: NIH training grant; Grant number: T32-GM007183; Grant sponsor: National Science Foundation; Grant number: DGE-1144082. *Correspondence to: Joseph Thornton, GCIS W504A, 929 E 57th St, Chicago, IL 60637. E-mail: 1204 PROTEIN SCIENCE 2016 VOL 25:1204—1218 fore not surprising that the functional effect of changing an amino acid often depends on the specific sequence of the protein into which the mutation is introduced. This dependency on genetic context has long been called epistasis by geneticists.1 Epistasis is invoked when the combined effect of two or more mutations deviates from that predicted by adding their individual effects. Although studies of epistasis have traditionally focused on genetic interactions between mutations at different loci,1 recent research has begun to address epistasis within proteins—its prevalence, C 2016 The Protein Society Published by Wiley-Blackwell. V biochemical mechanisms, and impacts on evolution. A consensus view of these subjects has not yet emerged however. Some papers conclude that epistasis is “rampant”2 or even the “primary factor” in protein evolution,3 whereas others claim that the frequency and magnitude of epistasis is “sufficiently low” such that it does not strongly affect the patterns of substitution in evolving proteins.4 There is also no clear picture of the mechanisms that cause epistasis: many papers have focused exclusively on epistasis mediated by effects on protein stability,5–9 although a few have addressed effects on protein conformation, ligand binding, and allostery.10–12 These disagreements reflect, at least in part, the lack of a unified discussion of the parallels and contrasts now emerging from the diverse modes of analysis applied to epistasis and its effects on protein evolution. Here we attempt such a unified view, focusing on the following specific questions: How important a factor is epistasis in changing the effects of mutations during the course of evolutionary history? Does epistasis typically amplify or dampen the effect of individual mutations? Does most evolutionarily relevant epistasis reflect very specific interactions between mutations—for example, with only one potential “permissive” mutation that can open the path for another specific mutation—or are many-to-one, one-to-many, or many-tomany interactions more common? What are the molecular mechanisms of interaction that produce each form of epistasis? And how do epistatic interactions of these various types influence the pathways and outcomes of long-term protein evolution? Epistasis and Protein Sequence Space The concept of sequence space provides a useful metaphor for understanding the relationship between a protein’s sequence, its physical or biological properties, and its evolution. Sequence space is a multidimensional representation of all possible protein genotypes, each connected to its neighbors by edges representing changes in a single residue.13 Assigning physical or biological properties to each genotype yields a “topological map” of the sequence space, just as a topological map of a geographic landscape assigns elevations to locations defined by their latitudinal and longitudinal coordinates. Epistasis makes the topology of sequence space “rugged”,14 in that the physical or biological effect of a mutation differs in sign or magnitude depending on the sequence background into which it is introduced; similarly, on a rugged geographical landscape, the change in elevation caused by a step in some direction varies dramatically depending on the starting point. As proteins evolve, they follow trajectories through sequence space, so this topology also determines how mutation, drift, selection, and other Starr and Thornton forces can drive genetic and functional evolution. A typical trajectory in natural or directed protein evolution consists of iterative mutational steps between functional proteins; trajectories involving strongly deleterious mutations are considered unlikely, because nonfunctional variants of biologically important proteins will usually reduce fitness and therefore be removed by natural selection.13,15–17 In the absence of epistasis, any mutation that changes protein properties in a beneficial way can be fixed by natural selection, irrespective of the genetic background in which it occurs; the result is a large number of passable trajectories through sequence space to the functional optimum that combines all of the beneficial sequence states. When epistasis is present, however, a mutation may be beneficial in some backgrounds but deleterious (or neutral) in others; the number of passable trajectories becomes smaller, the fixation of any one mutation may be contingent on the prior occurrence of other specific mutations, and there may be multiple local optima, consisting of mutually conditional beneficial states, isolated from each other by trajectories of low fitness. Epistasis can therefore affect evolutionary processes in dramatic ways. First, it can create a strong path-dependency in trajectories of protein evolution,18–21 because the mutations that are stochastically fixed may determine which functional optimum an evolving protein ultimately occupies, and these optima may differ not only in primary sequence but also in interesting physical or biological properties. Second, epistasis can yield evolutionary “dead-ends” in sequence space, from which a potentially beneficial mutation is not immediately accessible; in such cases, a relaxation of selection or even selection for other protein properties is necessary before a trajectory is opened to a superior optimum.10,22–28 Third, epistasis can cause a mutation that confers or improves a function in one protein to have no effect or even be strongly deleterious in a related protein;2,21,29 as a result, attempts to leverage natural sequence variation or experimental observations to predict mutational effects or engineer proteins with desired properties often fail.30 These issues highlight why characterizing epistasis—including the breadth of its effect, its mechanistic underpinnings, and its evolutionary impact—is important for our basic understanding of protein biochemistry and evolution. Prevalence and Strength of Epistasis How prevalent is epistasis within proteins, how strongly does it modulate the effects of mutations, and to what extent does this context-dependence affect long-term evolution? Studies of these questions have used two primary approaches—deep mutational scanning of large numbers of mutations in individual proteins, and analyses of changes in PROTEIN SCIENCE VOL 25:1204—1218 1205 mutational effects across long-term trajectories of protein evolution. Epistasis in a protein’s local sequence neighborhood A recently developed technique called deep mutational scanning makes it possible to characterize a very large library of mutant versions of some protein of interest with respect to some physical or biological property. By analyzing many or all variants that differ by one or two amino acids from a starting protein, it is possible to comprehensively characterize pairwise epistatic interactions in that protein’s local sequence neighborhood.31–35 In the absence of epistasis, the behavior of double mutants can be predicted with perfect accuracy by adding the effects of their constituent single mutations. (That is, R2 approaches 1 for the correlation between observed and predicted double mutant function.) In contrast, on a completely epistatic landscape, the effect of a mutation is completely independent in every background (so R2 approaches 0). Experiments reveal an intermediate prevalence of epistasis: the properties of single mutants predict double mutant behavior moderately well (R2  0.65–0.75).31–33 This result indicates that strong epistasis is not all-pervasive, pointing instead to epistasis that is pervasive and weak or relatively rare and strong. In fact, it appears that both types of interactions are important: a comprehensive study of pairwise interactions in protein G domain 1 (GB1) found strong deviations from additivity (by a factor >2) in 5% of all pairs of mutations, while weak epistasis (<2-fold deviation) affected 30% of pairs34 (see also Ref. 35). Thus, small-effect epistasis is very common; largeeffect epistasis is less pervasive but still affects a substantial number of mutations. Does epistasis tend to affect protein properties in one direction more than another? In “negative epistasis” a double mutant’s measured phenotype has a smaller value than expected under additivity [e.g., Fig. 1(C,D,G)], whereas in “positive epistasis” the phenotype is greater than predicted [Fig. 1(E,F,H)].1 In deep mutational scanning studies of ligand affinity and fitness effects, far more pairs exhibit negative than positive epistasis: the former group outnumbers the latter by a factor of 3–20.33–35 Most mutations have deleterious effects on these phenotypes, so negative epistasis in the majority of cases acts synergistically to make double mutants worse than either single mutant alone [Fig. 1(D)].33–35 This kind of epistasis would cause weakly deleterious mutations to become progressively less evolutionarily accessible as modifying mutations accumulate. Of particular importance for evolution is positive sign epistasis [Fig. 1(H)], in which a pair of deleterious or neutral mutations becomes beneficial 1206 PROTEINSCIENCE.ORG when combined. Although far less prevalent than negative epistasis, positive sign epistasis still appears to be widespread. In GB1, most mutations that are deleterious have at least one or more interacting mutations elsewhere in the protein that make it beneficial or neutral.34 Positive epistasis can open mutational trajectories to combinations of substitutions that would otherwise have been inaccessible. For example, in a high-throughput screen of a mutant protein library for variants that maintained the wild-type function, about 95% of the functional variants recovered would have been predicted to be nonfunctional from the effects of single mutations alone.36 These deep mutational scanning studies provide important insights into how epistasis might affect the first stages of an evolutionary process that begins from present-day forms, initially closing many paths to beneficial combinations but sometimes opening new ones. But the strategy leaves untouched important questions about the effect of epistasis on long-term historical protein evolution. There is plenty of epistasis in the local sequence neighborhood of a protein, but does this epistasis actually matter in determining proteins’ historical trajectories? For example, mutational scans suggest that many mutations manifest sign epistasis in their interactions, but how frequently does the direction of a mutation’s effect actually change during evolution? Is the strength and pervasiveness of epistasis in the immediate neighborhood of extant proteins similar to that in the much larger tracts of sequence space traversed by proteins evolving over hundreds of millions of years? Answering these questions requires direct analysis of epistasis across long-term trajectories of protein evolution. Epistasis in long-term protein evolution One way to gain insight into epistasis in real protein evolution is to compare the effects of some mutation on physical or biological properties when it is introduced into different proteins related by evolutionary descent (homologs). Some studies have addressed this question experimentally, while others have used computational approaches to indirectly infer the prevalence and strength of epistasis during longterm evolution. Experimental comparisons of mutational effects between homologs. Manipulative experiments on protein homologs point to both strong and pervasive effects of epistasis that cause the functional effects of mutations to differ between related proteins. One study tested the functional effect of 168 amino acid differences that separate orthologs enzymes that have maintained the same function in two bacterial species.2 Each individual residue from one ortholog was introduced into the other, and about one third Epistasis in Protein Evolution Figure 1. Patterns of epistasis between mutations. (A and B) Mutations a)A and b)B behave additively (non-epistatically) with respect to the measured phenotype (e.g., stability, fitness): the phenotypic effect of a state at one site is independent of the state of the other. (C and D) The two mutations exhibit negative epistasis: the double mutant AB has a lower measured phenotype than would be expected from the effects of A and B alone, regardless of the direction of the effect.1 (E and F) The two mutations exhibit positive epistasis: the double mutant AB has a greater phenotype than would be expected from the effects of A and B alone. (G) The two mutations exhibit negative sign epistasis: the sign of the phenotypic effect of state B depends on the state at site A. (H) The two mutations exhibit reciprocal sign epistasis: the sign of the phenotypic effect of either mutation changes depends on the state at the other. of these “sequence swaps” severely decreased enzyme activity. This result indicates that permissive epistatic interactions made the residue tolerable in its native background, that restrictive epistatic mutations made it intolerable in the other, or both. A similar study examined all combinations of nine variable residues that differ between closely related orthologs proteins and statistically determined both the average effects of each residue on catalytic activity, as well as the variance of its effect across different combinations.37 The standard deviation of every mutation’s effect was at least 45% of its average effect (up to 75% in the most extreme case), indicating significant epistasis among the sequence differences between the proteins. These studies demonstrate widespread epistasis, but they do not trace the accumulation of epistatically interacting mutations over time. A rapid study addressed this question using the rapid evolution of influenza nucleoprotein.7 The mutational trajectory of the protein over the last 39 years was reconstructed. Each of the substitutions that occurred during this trajectory was assessed for its effects on Starr and Thornton viral RNA transcription when introduced into the sequence context in which it occurred historically and into the sequence from an extinct strain that closely resembles an ancestral version of the protein. Every substitution was neutral in the background in which it occurred, but three were radically deleterious with respect to both function and fitness in the ancestral background, indicating relatively rare but extremely strong epistasis that allowed these mutations to be tolerated later. The above examples illuminate the variability in mutational effect for states that were actually incorporated into diverging proteins during evolution. But what is the impact of epistasis on the effects of all mutations, including those that are never observed because they are deleterious? A recent study compared site-specific mutational preferences between two influenza nucleoprotein orthologs by assessing the effect on viral fitness of all 19 possible single-amino-acid replacement mutations at every site in the two proteins, whether or not they changed during evolution.4 The two proteins differ at only 6% of sites, but significant differences in site-specific amino acid preference were found at 3– 15% percent of sites (depending on the statistical method used to evaluate differences). Thus, on average, each substitution during the evolution of these two closely related proteins modulated the amino acid preferences at one or two other sites. Strong epistasis is also apparent in laboratory evolution studies. One study placed a protein under strong selective pressure to evolve a new activity and then reimposed selection for the original activity, a trajectory that involved 28 amino acid changes in all.21 The “ancestral” amino acid state at each of these 28 sites was then introduced singly into the “derived” protein, and the derived states were each introduced into the ancestral protein to test for context-dependence. Almost half of the substitutions were deleterious when swapped into the other background, pointing to widespread epistatic interactions among the sites and states that were substituted during the laboratory evolutionary process. Comparative sequence analysis. Computational analyses of protein sequence data have investigated epistasis by seeking evidence that the effects of mutations differ among phylogenetic lineages. These studies have detected several major signatures (Fig. 2) that point to a strong and pervasive effect of epistasis on protein evolution. First, amino acid states that cause disease in one lineage frequently correspond to a wild-type state in the orthologous protein from other species [Fig. 2(A)].38–44 These states do not cause disease in the lineages in which they have fixed, so other lineage-specific substitutions must have modulated their effects. Remarkably, of the sequen ...
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Article critique of Epistasis in protein evolution
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Article critique of Epistasis in protein evolution

The purpose of the article Epistasis in protein evolution is to study mutation and
evolution by focusing on epistasis which affects proteins. There is a lack of unified perspective
on the effects of epistasis on protein evolution, and the authors are attempting to come up with a
unified view of protein evolution. Different studies have been conducted on epistasis, and they
have not arrived at a single conclusion, one study concludes that epistasis is rampant while
another claims it is low. The purpose of the authors is to come up with a unified conclusion on
the effects of epistasis in evolving proteins. To do this the auth...

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