The simplicity of protein sequence-function relationships

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Sept. 5, 2023

How complicated is the genetic architecture of proteins - set causal effects by which sequence determines function? High-order epistatic interactions among residues are thought to be pervasive, making a protein's function difficult predict or understand from its sequence. Most studies, however, used methods that overestimate epistasis, because they analyze relative designated reference causing measurement noise and small local idiosyncrasies propagate into pervasive high-order have not effectively accounted for global nonlinearity in sequence-function relationship. Here we present new reference-free method jointly estimates specific across entire genotype-phenotype map. This yields maximally efficient explanation more robust than existing noise, partial sampling, model misspecification. We reanalyze 20 combinatorial mutagenesis experiments diverse find additive pairwise effects, along with simple account limited dynamic range, explain median 96% total variance measured phenotypes (and >92% every case). Only tiny fraction genotypes strongly affected third- higher-order epistasis. Genetic also sparse: number terms required vast majority smaller many orders magnitude. The relationship most therefore far simpler previously thought, opening way tractable approaches characterize it.

Language: Английский

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Stepwise neofunctionalization of the NF-κB family member Rel during vertebrate evolution

Nature Immunology, Journal Year: 2025, Volume and Issue: 26(5), P. 760 - 774

Published: April 30, 2025

Adaptive immunity and the five vertebrate NF-κB family members first emerged in cartilaginous fish, suggesting that divergence helped to facilitate adaptive immunity. One specialized function of Rel protein macrophages is activation Il12b, which encodes a key regulator T cell development. We found Il12b exhibits much greater dependence than inducible innate genes macrophages, with unique dimers depending on heightened intrinsic DNA-binding affinity. Chromatin immunoprecipitation followed by sequencing experiments defined differential preferences genome-wide, X-ray crystallography revealed residue supports affinity dimers. Unexpectedly, this residue, dimers, portion promoter bound were largely restricted mammals. Our findings reveal major structural transitions an member one its target promoters at late stage evolution apparently contributed immunoregulatory rewiring mammalian species.

Language: Английский

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Protein sequence landscapes are not so simple: on reference-free versus reference-based inference

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 30, 2024

In a recent preprint, Park, Metzger, and Thornton reanalyze 20 empirical protein sequence-function landscapes using "reference-free analysis" (RFA) method they recently developed. They argue that these are simpler less epistatic than earlier work suggested, attribute the difference to limitations of methods used in original analyses landscapes, which claim more sensitive measurement noise, missing data, other artifacts. Here, we show claims incorrect. Instead, find RFA introduced by Park et al. is exactly equivalent reference-based least-squares analysis many (and also Hadamard-based approach implement). Because reanalyzed fact identical, different conclusions drawn instead reflect interpretations parameters describing inferred landscapes; do not support conclusion epistasis plays only small role landscapes.

Language: Английский

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Addressing epistasis in the design of protein function

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(34)

Published: Aug. 12, 2024

Mutations in protein active sites can dramatically improve function. The site, however, is densely packed and extremely sensitive to mutations. Therefore, some mutations may only be tolerated combination with others a phenomenon known as epistasis. Epistasis reduces the likelihood of obtaining improved functional variants slows natural lab evolutionary processes. Research has shed light on molecular origins epistasis its role shaping trajectories outcomes. In addition, sequence- AI-based strategies that infer epistatic relationships from mutational patterns or experimental evolution data have been used design variants. recent years, combinations such approaches atomistic calculations successfully predicted highly combinatorial sites. These were thousands active-site variants, demonstrating that, while our understanding remains incomplete, determinants are critical for accurate now sufficiently understood. We conclude space explored by expanded enhance activities discover new ones. Furthermore, opens way systematically exploring sequence structure impacts function, deepening control over activity.

Language: Английский

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Epistasis facilitates functional evolution in an ancient transcription factor

eLife, Journal Year: 2023, Volume and Issue: 12

Published: July 12, 2023

A protein's genetic architecture - the set of causal rules by which its sequence produces functions also determines possible evolutionary trajectories. Prior research has proposed that proteins is very complex, with pervasive epistatic interactions constrain evolution and make function difficult to predict from sequence. Most this work analyzed only direct paths between two interest excluding vast majority genotypes trajectories considered a single protein function, leaving unaddressed functional specificity impact on new functions. Here, we develop method based ordinal logistic regression directly characterize global determinants multiple 20-state combinatorial deep mutational scanning (DMS) experiments. We use it dissect transcription factor's for DNA, using data DMS an ancient steroid hormone receptor's capacity activate biologically relevant DNA elements. show recognition consists dense main pairwise effects involve virtually every amino acid state in protein-DNA interface, but higher-order epistasis plays tiny role. Pairwise enlarge sequences are primary different They massively expand number opportunities single-residue mutations switch one target another. By bringing variants close together space, therefore facilitates rather than constrains

Language: Английский

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Ancient biases in phenotype production drove the functional evolution of a protein family

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 29, 2025

ABSTRACT Biological systems may be biased in the phenotypes they can access by mutation 1–7 , but extent of these biases and their causal role evolution extant phenotypic diversity remains unclear. There are three major challenges: it is difficult to isolate effect bias genotype-phenotype (GP) map from that natural selection producing 6,8–11 universe possible genotypes so vast complex a direct characterization has been impossible, most evolved long ago species whose GP maps cannot recovered. Here we develop exhaustive multi-phenotype deep mutational scanning experimentally characterize complete two reconstructed ancestral steroid receptor proteins, which existed during an ancient phylogenetic interval when new phenotype—specific binding DNA response element—evolved 12 . We measured all specificity encoded amino acid combinations at sites protein’s interface. found structured strong global bias—unequal propensity encode various phenotypes—and extreme heterogeneity accessible around each genotype, strongly affect on both short timescales. Distinct steered toward lineage-specific functional history. Our findings establish relationship were factors evolutionary process produced present-day patterns conservation this protein family.

Language: Английский

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Gauge fixing for sequence-function relationships

PLoS Computational Biology, Journal Year: 2025, Volume and Issue: 21(3), P. e1012818 - e1012818

Published: March 20, 2025

Quantitative models of sequence-function relationships are ubiquitous in computational biology, e.g., for modeling the DNA binding transcription factors or fitness landscapes proteins. Interpreting these models, however, is complicated by fact that values model parameters can often be changed without affecting predictions. Before meaningfully interpreted, one must remove degrees freedom (called “gauge freedoms” physics) imposing additional constraints (a process called “fixing gauge”). However, strategies fixing gauge have received little attention. Here we derive an analytically tractable family gauges a large class relationships. These derived context with all-order interactions, but important subset applied to diverse types including additive pairwise-interaction and higher-order interactions. Many commonly used special cases within this family. We demonstrate utility showing how different choices both explore complex activity reveal simplified approximately correct localized regions sequence space. The results provide practical gauge-fixing exploration interpretation.

Language: Английский

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Computational and Experimental Exploration of Protein Fitness Landscapes: Navigating Smooth and Rugged Terrains

Biochemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 25, 2025

Proteins evolve through complex sequence spaces, with fitness landscapes serving as a conceptual framework that links to function. Fitness can be smooth, where multiple similarly accessible evolutionary paths are available, or rugged, the presence of local optima complicate evolution and prediction. Indeed, many proteins, especially those functions under selection pressures, exist on rugged landscapes. Here we discuss theoretical underpins our understanding landscapes, alongside recent work has advanced understanding─particularly biophysical basis for smoothness versus ruggedness. Finally, address rapid advances have been made in computational experimental exploration exploitation how these identify efficient routes protein optimization.

Language: Английский

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Gauge fixing for sequence-function relationships

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 13, 2024

Quantitative models of sequence-function relationships are ubiquitous in computational biology, e.g., for modeling the DNA binding transcription factors or fitness landscapes proteins. Interpreting these models, however, is complicated by fact that values model parameters can often be changed without affecting predictions. Before meaningfully interpreted, one must remove degrees freedom (called "gauge freedoms" physics) imposing additional constraints (a process called "fixing gauge"). However, strategies fixing gauge have received little attention. Here we derive an analytically tractable family gauges a large class relationships. These derived context with all-order interactions, but important subset applied to diverse types including additive pairwise-interaction and higher-order interactions. Many commonly used special cases within this family. We demonstrate utility showing how different choices both explore complex activity reveal simplified approximately correct localized regions sequence space. The results provide practical gauge-fixing exploration interpretation.

Language: Английский

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Specialization restricts the evolutionary paths available to yeast sugar transporters

Molecular Biology and Evolution, Journal Year: 2024, Volume and Issue: 41(11)

Published: Oct. 30, 2024

Abstract Functional innovation at the protein level is a key source of evolutionary novelties. The constraints on functional innovations are likely to be highly specific in different proteins, which shaped by their unique histories and extent global epistasis that arises from structures biochemistries. These contextual nuances sequence–function relationship have implications both for basic understanding process engineering proteins with desirable properties. Here, we investigated molecular basis novel function model member an ancient, conserved, biotechnologically relevant family. Major Facilitator Superfamily sugar porters functionally diverse group thought plastic evolvable. By dissecting recent α-glucoside transporter yeast Saccharomyces eubayanus, show ability transport substrate requires high-order interactions between many regions numerous residues proximal channel. To reconcile diversity this family constrained evolution protein, generated new, state-of-the-art genome annotations 332 Saccharomycotina species spanning ∼400 My evolution. integrating phylogenetic phenotypic analyses across these species, transporters evolved multifunctional ancestor became subfunctionalized. accumulation additive epistatic substitutions entrenched subfunction, made simultaneous acquisition multiple interacting only reasonably accessible path novelty.

Language: Английский

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