ABSTRACT
How individual enzymes evolved is relatively well understood. However, individual enzymes rarely confer a physiological advantage on their own. Judging by its current state, the emergence of metabolism seemingly demanded the simultaneous emergence of many enzymes. Indeed, how multicomponent interlocked systems, like metabolic pathways, evolved is largely an open question. This complexity can be unlocked if we assume that survival of the fittest applies not only to genes and enzymes but also to the metabolites they produce. This review develops our current knowledge of enzyme evolution into a wider hypothesis of pathway and network evolution. We describe the current models for pathway evolution and offer an integrative metabolite-enzyme coevolution hypothesis. Our hypothesis addresses the origins of new metabolites and of new enzymes and the order of their recruitment. We aim to not only survey established knowledge but also present open questions and potential ways of addressing them.
Subject(s)
Enzymes/genetics , Enzymes/metabolism , Evolution, Molecular , Metabolic Networks and Pathways/genetics , Enzymes/chemistry , Kinetics , Models, Biological , Models, Molecular , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Phylogeny , Substrate Specificity/geneticsABSTRACT
The rediscovery of Mendel's work showing that the heredity of phenotypes is controlled by discrete genes was followed by the reconciliation of Mendelian genetics with evolution by natural selection in the middle of the last century with the Modern Synthesis. In the past two decades, dramatic advances in genomic methods have facilitated the identification of the loci, genes, and even individual mutations that underlie phenotypic variants that are the putative targets of natural selection. Moreover, these methods have also changed how we can study adaptation by flipping the problem around, allowing us to first examine what loci show evidence of having been under selection, and then connecting these genetic variants to phenotypic variation. As a result, we now have an expanding list of actual genetic changes that underlie potentially adaptive phenotypic variation. Here, we synthesize how considering the effects of these adaptive loci in the context of cellular environments, genomes, organisms, and populations has provided new insights to the genetic architecture of adaptation.
Subject(s)
Adaptation, Physiological , Genetic Variation , Selection, Genetic , Adaptation, Physiological/genetics , Biological Evolution , Genetics , PhenotypeABSTRACT
Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits assemble with a precise stoichiometry and arrangement such that two chemically distinct agonist-binding sites are formed between specific subunit pairs. How this subunit complexity evolved and became entrenched is unclear. Here we show that a single historical amino acid substitution is able to constrain the subunit stoichiometry of functional acetylcholine receptors. Using a combination of ancestral sequence reconstruction, single-channel electrophysiology, and concatenated subunits, we reveal that an ancestral ß-subunit can not only replace the extant ß-subunit but can also supplant the neighboring δ-subunit. By forward evolving the ancestral ß-subunit with a single amino acid substitution, we restore the requirement for a δ-subunit for functional channels. These findings reveal that a single historical substitution necessitates an increase in acetylcholine receptor complexity and, more generally, that simple stepwise mutations can drive subunit entrenchment in this model heteromeric protein.
Subject(s)
Amino Acid Substitution , Protein Multimerization , Receptors, Nicotinic/genetics , Cell Line , Evolution, Molecular , Humans , Protein Binding , Protein Domains , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolismABSTRACT
Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Furthermore, duplication and promiscuity in such enzyme families limits their functional prediction, which has produced a burgeoning set of incompletely annotated genes in plant genomes. Here, we address these challenges using BAHD acyltransferases as a model. This fast-evolving family expanded drastically in land plants, increasing from one to five copies in algae to approximately 100 copies in diploid angiosperm genomes. Compilation of >160 published activities helped visualize the chemical space occupied by this family and define eight different classes based on structural similarities between acceptor substrates. Using orthologous groups (OGs) across 52 sequenced plant genomes, we developed a method to predict BAHD acceptor substrate class utilization as well as origins of individual BAHD OGs in plant evolution. This method was validated using six novel and 28 previously characterized enzymes and helped improve putative substrate class predictions for BAHDs in the tomato genome. Our results also revealed that while cuticular wax and lignin biosynthetic activities were more ancient, anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms. The OG-based analysis enabled identification of signature motifs in anthocyanin-acylating BAHDs, whose importance was validated via molecular dynamic simulations, site-directed mutagenesis and kinetic assays. Our results not only describe how BAHDs contributed to evolution of multiple chemical phenotypes in the plant world but also propose a biocuration-enabled approach for improved functional annotation of plant enzyme families.
Subject(s)
Acyltransferases , Solanum lycopersicum , Acyltransferases/metabolism , Anthocyanins/metabolism , Genome, Plant/genetics , Solanum lycopersicum/genetics , Phylogeny , Plants/metabolismABSTRACT
The genetic origins of novelty are of central interest in evolutionary biology. ISG15 and UBA7 are present only in vertebrates. The emergence and evolution of them are not clear. Phylogenetic comparisons revealed that UBA7 descends from gene duplication, and ISG15 and UBA7 arose from UBB/UBC and UBA1, respectively. Uba7 exhibits ubiquitin-activation activity in fish but not tetrapods, suggesting that the relationship of ISG15/Uba7 was promiscuous in origin but was later coopted toward higher specificity. Zebrafish Uba7 is capable of activating the ubiquitin cascade in vitro and in vivo, and it displays distinct specificity preference toward substrates and E2 enzymes compared to zebrafish Uba1. These results together provide a framework for understanding the origin and diversification of ISG15/Uba7 and may serve as a paradigmatic example in which an originally minor functionality in an old gene is made into a new high-specificity protein through random mutations and natural selection.
Subject(s)
Ubiquitins , Zebrafish , Animals , Cytokines/genetics , Cytokines/metabolism , Phylogeny , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitins/genetics , Ubiquitins/metabolism , Zebrafish/geneticsABSTRACT
Polymorphism in the alcohol dehydrogenase (ADH) protein of Drosophila melanogaster, like genetic variation in many other enzymes, has long been hypothesized to be maintained by a selective trade-off between thermostability and enzyme activity. Two major Adh variants, named Fast and Slow, are distributed along latitudinal clines on several continents. The balancing selection trade-off hypothesis posits that Fast is favored at high latitudes because it metabolizes alcohol faster, whereas Slow is favored at low latitudes because it is more stable at high temperatures. Here we use biochemical and physiological assays of precisely engineered genetic variants to directly test this hypothesis. As predicted, the Fast protein has higher catalytic activity than Slow, and both the Fast protein and regulatory variants linked to it confer greater ethanol tolerance on transgenic animals. But we found no evidence of a temperature-mediated trade-off: The Fast protein is not less stable or active at high temperatures, and Fast alleles increase ethanol tolerance and survivorship at all temperatures tested. Further, analysis of a population genomic dataset reveals no signature of balancing selection in the Adh gene. These results provide strong evidence against balancing selection driven by a stability/activity trade-off in Adh, and they justify caution about this hypothesis for other enzymes except those for which it has been directly tested. Our findings tentatively suggest that environment-specific selection for the Fast allele, coupled with demographic history, may have produced the observed pattern of Adh variation.
Subject(s)
Alcohol Dehydrogenase/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Ethanol/metabolism , Polymorphism, Genetic/genetics , Alleles , Animals , Animals, Genetically Modified , TemperatureABSTRACT
Metabolic networks are complex cellular systems dependent on the interactions among, and regulation of, the enzymes in the network. Although there is great diversity of types of enzymes that make up metabolic networks, the models meant to understand the possible evolutionary outcomes following duplication neglect specifics about the enzyme, pathway context, and cellular constraints. To illuminate the mechanisms that shape the evolution of biochemical pathways, I functionally characterize the consequences of gene duplication of an enzyme family that performs multiple subsequent enzymatic reactions (a multistep enzyme) in the corticosteroid pathway in primates. The products of the corticosteroid pathway (aldosterone and cortisol) are steroid hormones that regulate metabolism and stress response in tetrapods. These steroid hormones are synthesized by a multistep enzyme Cytochrome P450 11B (CYP11B) that performs subsequent steps on different carbon atoms of the steroid derivatives. Through ancestral state reconstruction and in vitro characterization, I find that the primate ancestor of the CYP11B1 and CYP11B2 paralogs had moderate ability to synthesize both cortisol and aldosterone. Following duplication in Old World primates, the CYP11B1 homolog specialized on the production of cortisol, whereas its paralog, CYP11B2, maintained its ability to perform multiple subsequent steps as in the ancestral pathway. Unlike CYP11B1, CYP11B2 could not specialize on the production of aldosterone because it is constrained to perform earlier steps in the corticosteroid synthesis pathway to achieve the final product aldosterone. These results suggest that enzyme function, pathway context, along with tissue-specific regulation, both play a role in shaping potential outcomes of metabolic network elaboration.
Subject(s)
Adrenal Cortex Hormones/biosynthesis , Evolution, Molecular , Gene Duplication , Primates/genetics , Steroid 11-beta-Hydroxylase/genetics , Animals , Humans , Primates/metabolism , Steroid 11-beta-Hydroxylase/metabolismABSTRACT
Global functions of nicotinic acetylcholine receptors, such as subunit cooperativity and compatibility, likely emerge from a network of amino acid residues distributed across the entire pentameric complex. Identification of such networks has stymied traditional approaches to acetylcholine receptor structure and function, likely due to the cryptic interdependency of their underlying amino acid residues. An emerging evolutionary biochemistry approach, which traces the evolutionary history of acetylcholine receptor subunits, allows for rational mapping of acetylcholine receptor sequence space, and offers new hope for uncovering the amino acid origins of these enigmatic properties.
Subject(s)
Evolution, Molecular , Receptors, Cholinergic/chemistry , Animals , Humans , Protein Structure, Tertiary , Receptors, Cholinergic/metabolism , Structure-Activity RelationshipABSTRACT
Mutations in the chloroquine resistance transporter (PfCRT) are the primary determinant of chloroquine (CQ) resistance in the malaria parasite Plasmodium falciparum. A number of distinct PfCRT haplotypes, containing between 4 and 10 mutations, have given rise to CQ resistance in different parts of the world. Here we present a detailed molecular analysis of the number of mutations (and the order of addition) required to confer CQ transport activity upon the PfCRT as well as a kinetic characterization of diverse forms of PfCRT. We measured the ability of more than 100 variants of PfCRT to transport CQ when expressed at the surface of Xenopus laevis oocytes. Multiple mutational pathways led to saturable CQ transport via PfCRT, but these could be separated into two main lineages. Moreover, the attainment of full activity followed a rigid process in which mutations had to be added in a specific order to avoid reductions in CQ transport activity. A minimum of two mutations sufficed for (low) CQ transport activity, and as few as four conferred full activity. The finding that diverse PfCRT variants are all limited in their capacity to transport CQ suggests that resistance could be overcome by reoptimizing the CQ dosage.
Subject(s)
Chloroquine/metabolism , Drug Resistance , Malaria, Falciparum/metabolism , Membrane Transport Proteins/genetics , Mutation/genetics , Parasites/metabolism , Plasmodium falciparum/metabolism , Protozoan Proteins/genetics , Amino Acid Sequence , Animals , Biological Transport , Haplotypes , Kinetics , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Oocytes , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Recombinant Proteins/metabolism , Structure-Activity Relationship , Transfection , Xenopus laevisABSTRACT
The genetic and biophysical mechanisms by which new protein functions evolve is a central question in evolutionary biology, biochemistry, and biophysics. Of particular interest is whether major shifts in protein function are caused by a few mutations of large effect and, if they are, the mechanisms that mediate these changes. Here we combine ancestral protein reconstruction with genetic manipulation and explicit studies of protein structure and dynamics to dissect an ancient and discrete shift in ligand specificity in the steroid receptors, a family of biologically essential hormone-controlled transcription factors. We previously found that the ancestor of the entire steroid receptor family was highly specific for estrogens, but its immediate phylogenetic descendant was sensitive only to androgens, progestogens, and corticosteroids. Here we show that this shift in function was driven primarily by two historical amino acid changes, which caused a â¼70,000-fold change in the ancestral protein's specificity. These replacements subtly changed the chemistry of two amino acids, but they dramatically reduced estrogen sensitivity by introducing an excess of interaction partners into the receptor/estrogen complex, inducing a frustrated ensemble of suboptimal hydrogen bond networks unique to estrogens. This work shows how the protein's architecture and dynamics shaped its evolution, amplifying a few biochemically subtle mutations into major shifts in the energetics and function of the protein.
Subject(s)
Biophysics , Evolution, Molecular , Mutation , Receptors, Steroid/genetics , Ligands , Models, Molecular , Phylogeny , Protein Binding , Receptors, Steroid/chemistry , Receptors, Steroid/metabolismABSTRACT
Magnetic sense, or termed magnetoreception, has evolved in a broad range of taxa within the animal kingdom to facilitate orientation and navigation. MagRs, highly conserved A-type iron-sulfur proteins, are widely distributed across all phyla and play essential roles in both magnetoreception and iron-sulfur cluster biogenesis. However, the evolutionary origins and functional diversification of MagRs from their prokaryotic ancestor remain unclear. In this study, MagR sequences from 131 species, ranging from bacteria to humans, were selected for analysis, with 23 representative sequences covering species from prokaryotes to Mollusca, Arthropoda, Osteichthyes, Reptilia, Aves, and mammals chosen for protein expression and purification. Biochemical studies revealed a gradual increase in total iron content in MagRs during evolution. Three types of MagRs were identified, each with distinct iron and/or iron-sulfur cluster binding capacity and protein stability, indicating continuous expansion of the functional roles of MagRs during speciation and evolution. This evolutionary biochemical study provides valuable insights into how evolution shapes the physical and chemical properties of biological molecules such as MagRs and how these properties influence the evolutionary trajectories of MagRs.
Subject(s)
Iron-Sulfur Proteins , Animals , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/chemistry , Biological Evolution , Evolution, Molecular , Phylogeny , Iron/metabolismABSTRACT
A protein's sequence determines its conformational energy landscape. This, in turn, determines the protein's function. Understanding the evolution of new protein functions therefore requires understanding how mutations alter the protein energy landscape. Ancestral sequence reconstruction (ASR) has proven a valuable tool for tackling this problem. In ASR, one phylogenetically infers the sequences of ancient proteins, allowing characterization of their properties. When coupled to biophysical, biochemical, and functional characterization, ASR can reveal how historical mutations altered the energy landscape of ancient proteins, allowing the evolution of enzyme activity, altered conformations, binding specificity, oligomerization, and many other protein features. In this article, we review how ASR studies have been used to dissect the evolution of energy landscapes. We also discuss ASR studies that reveal how energy landscapes have shaped protein evolution. Finally, we propose that thinking about evolution from the perspective of an energy landscape can improve how we approach and interpret ASR studies.
Subject(s)
Evolution, Molecular , Proteins , Proteins/chemistry , Proteins/genetics , Proteins/metabolism , Humans , Protein Conformation , Thermodynamics , Animals , PhylogenyABSTRACT
Ancestral sequence reconstruction (ASR) is a powerful tool to study the evolution of proteins and thus gain deep insight into the relationships among protein sequence, structure, and function. A major barrier to its broad use is the complexity of the task: it requires multiple software packages, complex file manipulations, and expert phylogenetic knowledge. Here we introduce topiary, a software pipeline that aims to overcome this barrier. To use topiary, users prepare a spreadsheet with a handful of sequences. Topiary then: (1) Infers the taxonomic scope for the ASR study and finds relevant sequences by BLAST; (2) Does taxonomically informed sequence quality control and redundancy reduction; (3) Constructs a multiple sequence alignment; (4) Generates a maximum-likelihood gene tree; (5) Reconciles the gene tree to the species tree; (6) Reconstructs ancestral amino acid sequences; and (7) Determines branch supports. The pipeline returns annotated evolutionary trees, spreadsheets with sequences, and graphical summaries of ancestor quality. This is achieved by integrating modern phylogenetics software (Muscle5, RAxML-NG, GeneRax, and PastML) with online databases (NCBI and the Open Tree of Life). In this paper, we introduce non-expert readers to the steps required for ASR, describe the specific design choices made in topiary, provide a detailed protocol for users, and then validate the pipeline using datasets from a broad collection of protein families. Topiary is freely available for download: https://github.com/harmslab/topiary.
Subject(s)
Proteins , Software , Phylogeny , Amino Acid Sequence , Proteins/genetics , Proteins/chemistry , Sequence Alignment , Evolution, MolecularABSTRACT
We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.
Subject(s)
Oxidoreductases , Tetrahydrofolate Dehydrogenase , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/chemistry , Tetrahydrofolate Dehydrogenase/metabolism , Anti-Bacterial Agents , Drug Resistance, BacterialABSTRACT
The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always followed the same set of strategies for performing these critical tasks. Today, microbes access atmospheric sources of bioessential nitrogen through the activities of just one family of enzymes, nitrogenases. Here, we show that the only dinitrogen reduction mechanism known to date is an ancient feature conserved from nitrogenase ancestors. We designed a paleomolecular engineering approach wherein ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, Azotobacter vinelandii, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. Nitrogenase ancestors are active and robust to variable incorporation of one or more ancestral protein subunits. Further, we find that all ancestors exhibit the reversible enzymatic mechanism for dinitrogen reduction, specifically evidenced by hydrogen inhibition, which is also exhibited by extant A. vinelandii nitrogenase isozymes. Our results suggest that life may have been constrained in its sampling of protein sequence space to catalyze one of the most energetically challenging biochemical reactions in nature. The experimental framework established here is essential for probing how nitrogenase functionality has been shaped within a dynamic, cellular context to sustain a globally consequential metabolism.
Subject(s)
Azotobacter vinelandii , Nitrogenase , Nitrogenase/chemistry , Nitrogenase/genetics , Nitrogenase/metabolism , Nitrogen Fixation , Azotobacter vinelandii/genetics , Azotobacter vinelandii/metabolism , Amino Acid Sequence , Nitrogen/metabolismABSTRACT
Ancestral Sequence Reconstruction (ASR) allows one to infer the sequences of extinct proteins using the phylogeny of extant proteins. It consists of disclosing the evolutionary history-i.e., the phylogeny-of a protein family of interest and then inferring the sequences of its ancestors-i.e., the nodes in the phylogeny. Assisted by gene synthesis, the selected ancestors can be resurrected in the lab and experimentally characterized. The crucial step to succeed with ASR is starting from a reliable phylogeny. At the same time, it is of the utmost importance to have a clear idea on the evolutionary history of the family under study and the events that influenced it. This allows us to implement ASR with well-defined hypotheses and to apply the appropriate experimental methods. In the last years, ASR has become popular to test hypotheses about the origin of functionalities, changes in activities, understanding physicochemical properties of proteins, among others. In this context, the aim of this chapter is to present the ASR approach applied to the reconstruction of enzymes-i.e., proteins with catalytic roles. The spirit of this contribution is to provide a basic, hands-to-work guide for biochemists and biologists who are unfamiliar with molecular phylogenetics.
Subject(s)
Evolution, Molecular , Genetic Techniques , Phylogeny , Proteins/genetics , Sequence AnalysisABSTRACT
Cold temperature is prevalent across the biosphere and slows the rates of chemical reactions. Increased catalysis has been predicted to be a dominant adaptive trait of enzymes to reduced temperature, and this expectation has informed physical models for enzyme catalysis and influenced bioprospecting strategies. To systematically test rate enhancement as an adaptive trait to cold, we paired kinetic constants of 2223 enzyme reactions with their organism's optimal growth temperature (TGrowth) and analyzed trends of rate constants as a function of TGrowth. These data do not support a general increase in rate enhancement in cold adaptation. In the model enzyme ketosteroid isomerase (KSI), there is prior evidence for temperature adaptation from a change in an active site residue that results in a tradeoff between activity and stability. Nevertheless, we found that little of the rate constant variation for 20 KSI variants was accounted for by TGrowth. In contrast, and consistent with prior expectations, we observed a correlation between stability and TGrowth across 433 proteins. These results suggest that temperature exerts a weaker selection pressure on enzyme rate constants than stability and that evolutionary forces other than temperature are responsible for the majority of enzymatic rate constant variation.
Subject(s)
Adaptation, Physiological/physiology , Cold Temperature , Enzymes/chemistry , CatalysisABSTRACT
Epistasis occurs when the combined effect of two or more mutations differs from the sum of their individual effects, and reflects molecular interactions that affect the function and fitness of a protein. Epistasis is widely recognized as a key phenomenon that drives the dynamics of evolution. It can profoundly affect our ability to understand sequence-structure-function relationships, and thus has important implications for protein engineering and design. Characterizing higher-order epistasis, i.e., interactions between three or more mutations, can unveil hidden intramolecular interaction networks that underlie essential protein functions and their evolution. For this chapter, we developed an analytical pipeline that can standardize the study of intramolecular epistasis. We describe the generation and characterization of a combinatorial library, the statistical analysis of mutational epistasis, and finally, the depiction of epistatic networks on the 3D structure of a protein. We anticipate that this pipeline will benefit the increasing number of scientists that are interested in the functional characterization of mutational libraries to provide a deeper understanding of the molecular mechanisms of protein evolution.
Subject(s)
Epistasis, Genetic , Evolution, Molecular , Mutation , Proteins/genetics , Proteins/metabolismABSTRACT
Ancestral sequence reconstruction (ASR) is a powerful tool to infer primordial sequences from contemporary, i.e., extant ones. An essential element of ASR is the computation of a phylogenetic tree whose leaves are the chosen extant sequences. Most often, the reconstructed sequence related to the root of this tree is of greatest interest: It represents the common ancestor (CA) of the sequences under study. If this sequence encodes a protein, one can "resurrect" the CA by means of gene synthesis technology and study biochemical properties of this extinct predecessor with the help of wet-lab experiments.However, ASR deduces also sequences for all internal nodes of the tree, and the well-considered analysis of these "intermediates" can help to elucidate evolutionary processes. Moreover, one can identify key mutations that alter proteins or protein complexes and are responsible for the differing properties of extant proteins. As an illustrative example, we describe the protocol for the rapid identification of hotspots determining the binding of the two subunits within the heteromeric complex imidazole glycerol phosphate synthase.
Subject(s)
Evolution, Molecular , Proteins/metabolism , Algorithms , Biological Evolution , Phylogeny , Protein Binding , Proteins/classification , Proteins/geneticsABSTRACT
A central goal in biochemistry is to explain the causes of protein sequence, structure, and function. Mainstream approaches seek to rationalize sequence and structure in terms of their effects on function and to identify function's underlying determinants by comparing related proteins to each other. Although productive, both strategies suffer from intrinsic limitations that have left important aspects of many proteins unexplained. These limits can be overcome by reconstructing ancient proteins, experimentally characterizing their properties, and retracing their evolution through time. This approach has proven to be a powerful means for discovering how historical changes in sequence produced the functions, structures, and other physical/chemical characteristics of modern proteins. It has also illuminated whether protein features evolved because of functional optimization, historical constraint, or blind chance. Here we review recent studies employing ancestral protein reconstruction and show how they have produced new knowledge not only of molecular evolutionary processes but also of the underlying determinants of modern proteins' physical, chemical, and biological properties.