Emergence of polyproline II-like structure at early stages of experimental evolution from random polypeptides

To examine whether a primordial functional protein at the early stages of evolution has structural features, we carried out experimental evolution consisting of 25 cycles (generations) of mutation and selection toward DNA-binding function using a random-sequence polypeptide of 139 amino acid residues with no secondary structure as the initial sequence. In each generation, 16 clones were sampled arbitrarily for sequence analysis, and a phylogenetic tree was constructed. Polypeptide evolution proceeded from the initial point on branch I in 2 main directions of branches II and III. The initial and 2 evolved polypeptides (one at the 24th generation on branch III and the other at the 25th generation on branch II) were purified to examine their functional and structural properties. Although binding of the initial polypeptide to the target DNA was not detected by surface plasmon resonance measurements, the 2 evolved polypeptides bound to the DNA with dissociation constants of 1.6 and 1.0 microM, respectively, indicating an increase in affinity during the experimental evolution. Circular dichroism spectra of the evolved polypeptides, but not of the initial polypeptide, showed features characteristic of the polyproline II (PPII)-like structure, a left-handed helical structure commonly found in natural proteins, suggesting that the structure emerged through the experimental evolution. The same structural feature was found in another experimental evolution toward catalytic activity. These results demonstrate that the PPII-like structure is one of the common features that could have appeared in the early evolutionary stages of primordial functional protein.     

To Investigate Protein Evolution by Detecting Suppressed Epitope Structures

Material remains of ancestor nucleotides and proteins are largely unavailable, thus sequence comparison among homologous genes in present-day organisms forms the core of current knowledge of molecular evolution. Variation in protein three-dimensional structure is a basis for functional diversity. To study the evolution of three-dimensional structures in related proteins would significantly improve our understanding of protein evolution and function. A protein may contain ancestor conformations that have been allosterically suppressed by evolutionarily additive structures. Using monoclonal antibody probes to detect such conformation in proteins after removing the suppressor structure, our study demonstrates three-dimensional structure evidence for the evolutionary relationship between troponin I and troponin T, two subunits of the troponin complex in the Ca²⁺-regulatory system of striated muscle, and among their muscle type-specific isoforms. The experimental data show the feasibility of detecting evolutionarily suppressed history-telling structural states in proteins by removing conformational modulator segments added during evolution. In addition to identifying structural modifications that were critical to the emergence of diverged proteins, investigating this novel mode of evolution will help us to understand the origin and functional potential of protein structures.     

Constructal Law of Vascular Trees for Facilitation of Flow

Diverse tree structures such as blood vessels, branches of a tree and river basins exist in nature. The constructal law states that the evolution of flow structures in nature has a tendency to facilitate flow. This study suggests a theoretical basis for evaluation of flow facilitation within vascular structure from the perspective of evolution. A novel evolution parameter (Ev) is proposed to quantify the flow capacity of vascular structures. Ev is defined as the ratio of the flow conductance of an evolving structure (configuration with imperfection) to the flow conductance of structure with least imperfection. Attaining higher Ev enables the structure to expedite flow circulation with less energy dissipation. For both Newtonian and non-Newtonian fluids, the evolution parameter was developed as a function of geometrical shape factors in laminar and turbulent fully developed flows. It was found that the non-Newtonian or Newtonian behavior of fluid as well as flow behavior such as laminar or turbulent behavior affects the evolution parameter. Using measured vascular morphometric data of various organs and species, the evolution parameter was calculated. The evolution parameter of the tree structures in biological systems was found to be in the range of 0.95 to 1. The conclusion is that various organs in various species have high capacity to facilitate flow within their respective vascular structures.     

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

The acquisition of function is often associated with destabilizing mutations, giving rise to the stability–function tradeoff hypothesis. To test whether function is also accommodated at the expense of foldability, fibroblast growth factor‐1 (FGF‐1) was subjected to a comprehensive φ‐value analysis at each of the 11 turn regions. FGF‐1, a β‐trefoil fold, represents an excellent model system with which to evaluate the influence of function on foldability: because of its threefold symmetric structure, analysis of FGF‐1 allows for direct comparisons between symmetry‐related regions of the protein that are associated with function to those that are not; thus, a structural basis for regions of foldability can potentially be identified. The resulting φ‐value distribution of FGF‐1 is highly polarized, with the majority of positions described as either folded‐like or denatured‐like in the folding transition state. Regions important for folding are shown to be asymmetrically distributed within the protein architecture; furthermore, regions associated with function (i.e., heparin‐binding affinity and receptor‐binding affinity) are localized to regions of the protein that fold after barrier crossing (late in the folding pathway). These results provide experimental support for the foldability–function tradeoff hypothesis in the evolution of FGF‐1. Notably, the results identify the potential for folding redundancy in symmetric protein architecture with important implications for protein evolution and design.     

Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

The CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized.     

Statistical Limitations on Molecular Evolution

Abstract

Complexity of functions evolving in an evolution process are expected to be limited by the time length of an evolution process among other factors. This paper outlines a general method of deriving function-complexity limitations based on mathematical statistics and independent from details of a biological or genetic mechanism of the evolution of the function. Limitations on the emergence of life are derived, these limitations indicate a possibility of a very fast evolution and are consistent with “RNA world” hypothesis. The discussed method is general and can be used to characterize evolution of more specific biological organism functions and relate functions to genetic structures. The derived general limitations indicate that a co-evolution of multiple functions and species could be a slow process, whereas an evolution of a specific function might proceed very fast, so that no trace of intermediate forms (species) is preserved in fossil records of phenotype or DNA structure; this is consistent with a picture of “punctuated equilibrium”.     

Correlated mutations and residue contacts in proteins

The maintenance of protein function and structure constrains the evolution of amino acid sequences. This fact can be exploited to interpret correlated mutations observed in a sequence family as an indication of probable physical contact in three dimensions. Here we present a simple and general method to analyze correlations in mutational behavior between different positions in a multiple sequence alignment. We then use these correlations to predict contact maps for each of 11 protein families and compare the result with the contacts determined by crystallography. For the most strongly correlated residue pairs predicted to be in contact, the prediction accuracy ranges from 37 to 68% and the improvement ratio relative to a random prediction from 1.4 to 5.1. Predicted contact maps can be used as input for the calculation of protein tertiary structure, either from sequence information alone or in combination with experimental information. © 1994 John Wiley & Sons, Inc.     

Capturing, sharing and analysing biophysical data from protein engineering and protein characterization studies

Large amounts of data are being generated annually on the connection between the sequence, structure and function of proteins using site-directed mutagenesis, protein design and directed evolution techniques. These data provide the fundamental building blocks for our understanding of protein function, molecular biology and living organisms in general. However, much experimental data are never deposited in databases and is thus ‘lost’ in journal publications or in PhD theses. At the same time theoretical scientists are in need of large amounts of experimental data for benchmarking and calibrating novel predictive algorithms, and theoretical progress is therefore often hampered by the lack of suitable data to validate or disprove a theoretical assumption. We present PEAT (Protein Engineering Analysis Tool), an application that integrates data deposition, storage and analysis for researchers carrying out protein engineering projects or biophysical characterization of proteins. PEAT contains modules for DNA sequence manipulation, primer design, fitting of biophysical characterization data (enzyme kinetics, circular dichroism spectroscopy, NMR titration data, etc.), and facilitates sharing of experimental data and analyses for a typical university-based research group. PEAT is freely available to academic researchers at http://enzyme.ucd.ie/PEAT.     

Structure evolution and incomplete induction

Evolutionary strategies such as the evolution strategy (Rechenberg 1965, 1973; Schwefel 1977) or genetic algorithms (Holland 1975; Goldberg 1989) have been widely applied to systems where parameters have to be determined according to a particular objective function. A necessary demand in all these experiments is that the structures of the objects to be optimised are well defined, because these structures are part of the objective function. With structure evolution the range of applications of evolutionary algorithms can now be expanded to tasks which are less accurately described, i.e. where the structures of the objects are fairly unknown. Heuristical effort is reduced first to defining structure components by combinations of which the structure space is generated. The structure space can be nearly infinitely large. Furthermore, the mutation procedures for structures have to be determined, complying with the demand for strong causality. In its computer model the algorithm of structure evolution involves the phenomenon of isolation, a feature of biological evolution additional to replication, mutation, and selection, which have already been implemented in other strategies. The idea of structure evolution is to let different but some what similar structures of an object compete in temporarily isolated populations where the respective parameter evolution is carried out. Thus structure evolution can perform a most effective search, both in structure and parameter space. The algorithm is demonstrated with two examples: a neural filter in a visual system and the topologies of frameworks. The first of the examples touches the problem of incompletely described tasks, and this paper will show that the effect of overlearning can be avoided by a learning procedure called incomplete induction, which fits best with the algorithm of structure evolution.     

The evolution of calcium biochemistry

The role of calcium in evolution is best understood from a perspective based on its intrinsic value as a divalent cation able to bind and precipitate inorganic and organic anions rapidly. This binding can be useful or inhibitory. Now treatment of binding or precipitation has two different interests in biological cells. The first is thermodynamic, that is the stress is on systems biology and the second is structure, that is molecular biology. In evolution both have equal weight being connected through exchange. This paper outlines first the systems biology of the evolution of calcium functions from prokaryotes to animals with brains. The calcium ion was the only good available candidate in the environment for the functions it performs. The second section of the paper describes the evolution of the proteins which allow the messenger function. We have discussed elsewhere the structure/function relationships of the proteins. Overall the evolving and increasing involvement of calcium as possibly the major control messenger of events outside cells to action inside them is an inevitable feature of the nature of ecological, that is environmental/organism, evolution.     

To the Final Goal: Can We Predict and Suggest Mutations for Protein to Develop Desired Phenotype?

Directed evolution of proteins is a good approach to develop desired phenotypes from existing proteins. Fully experimental protein evolution usually utilizes randomization of a given protein sequence by error-prone PCR or gene shuffling followed by high-throughput selection or timeconsuming screening method. However, these random methods create mutant library full of deleterious mutations. In addition, they need high-throughput screening or selection method to search for positive clones from an enormous size of mutant library. Construction of a mutant library while retaining the original function is important for efficient protein evolution because it greatly reduces time and effort for the identification of positive mutants. Therefore, researchers have tried to reduce the size of mutant library by minimizing the occurrence of deleterious mutants. Such efforts have led to the creation of a concept of ‘small but smart library’. For this goal, neutral drift theory has been applied. Although smart library greatly reduces the library size, it is still the beyond the capacity of low-throughput assay. In parallel, computational analysis of protein structure and efforts to discriminate mutatable residues from all residues of a given protein have been consistently pursued. Accumulated knowledge of protein evolution through random mutation and selection has improved our understanding of functions of amino acids in protein structure. Protein evolution by rational design is being developed based on such understanding. In this review, we describe how the use of semi-rationally designed library rather than completely random one has impacted the overall procedure of directed evolution. We also describe efforts made to evaluate the effect of single mutation. Such efforts will bring lazy boys to the final goal - computational mutation suggestion system.     

Statistical limitations on molecular evolution

Complexity of functions evolving in an evolution process are expected to be limited by the time length of an evolution process among other factors. This paper outlines a general method of deriving function-complexity limitations based on mathematical statistics and independent from details of a biological or genetic mechanism of the evolution of the function. Limitations on the emergence of life are derived, these limitations indicate a possibility of a very fast evolution and are consistent with "RNA world" hypothesis. The discussed method is general and can be used to characterize evolution of more specific biological organism functions and relate functions to genetic structures. The derived general limitations indicate that a co-evolution of multiple functions and species could be a slow process, whereas an evolution of a specific function might proceed very fast, so that no trace of intermediate forms (species) is preserved in fossil records of phenotype or DNA structure; this is consistent with a picture of "punctuated equilibrium".     

Peptide self-aggregation and peptide complementarity as bases for the evolution of peptide receptors: a review

This paper reviews the three major theories of peptide receptor evolution: (1) Dwyer's theory that peptide receptors evolved from self-aggregating peptides; (2) Root-Bernstein's theory that peptide receptors evolved from functionally and structurally complementary peptides; and (3) Blalock's theory that receptors evolved from hydropathically complementary sequences encoded in the antisense strand of the DNA encoding each peptide. The evidence to date suggests that the co-yevolution of peptides and their receptors is strongly constrained by one or more of these physicochemically based mechanisms, which argues against a random or frozen accident' model. The data also suggest that structure and function are integrally related from the earliest steps of receptor-ligand evolution so that peptide functionality is non-random and highly conserved in its origin. The result is a molecular paleontology' that reveals the evolutionary constraints that shaped the interaction of structure and function.     

Experimental studies of pollen carryover: Hummingbirds and Ipomopsis aggregata

Summary We present results of experiments designed to identify floral characteristics that influence patterns of pollen carryover by hummingbirds visiting Ipomopsis aggregata flowers. We used fluorescent dye powders as pollen analogues. For all four experimental treatments considered, amounts of dye deposited on recipient stigmas declined linearly as a function of flower position in a visitation sequence. The decline was significantly steeper when recipient flowers had pollen-carrying anthers than when they did not; whereas degree of stigma clogging and presence or absence of empty anthers did not influence carryover. From this we conclude that presence of pollen on recipient flowers significantly reduces the average number of subsequent flowers reached by donor pollen. We discuss mechanisms for this effect and its significance for the evolution of floral structure.     

Correlated Flexible Molecular Coding and Molecular Evolvability

Evolvability of biopolymers is based on molecular coding. The molecular coding is represented by biopolymer function vs monomeric sequence relationship, that is, a proper fitness landscape on the sequence space. On the other hand, molecular coding is mostly realized by monomeric sequence vs biopolymer structure relationship. We suggest the evolution of evolvability based on flexible or multiplex coding originating from flexible or polymorphic conformation of evolving biopolymers. We report a finding supporting that the amino acid landscape of the standard genetic code for an amino acid property which is more important to the protein function gives higher value of an evolvability measure. We developed a promising molecular construct which realized genotype-phenotype linking in order to study the in vitroprotein evolution to clarify above mentioned protein evolvability.     

Floral evolution in the Annonaceae: hypotheses of homeotic mutations and functional convergence

The recent publication of hypotheses explaining the homeotic control of floral organ identity together with the availability of increasingly comprehensive and well‐resolved molecular phylogenies presents an ideal opportunity for reassessing current knowledge of floral diversity and evolution in the Annonaceae. This review summarizes currently available information on selected aspects of floral structure and function, including: changes in the number of perianth whorls and the number of perianth parts per whorl; the evolution of sympetaly; the diversity and evolution of pollination chambers (with a novel classification of seven main structural forms of floral chamber based on the different arrangement, size and shape of petals); the evolution of perianth glands; floral unisexuality and hypotheses explaining the unexpectedly high frequency of occurrence of androdioecy; the origin and possible function of inner and outer staminodes; the evolution of stamen connective diversity and theca septation; and the origin of ‘true’ syncarpy and functionally equivalent extragynoecial compita. In each case, current ideas on the origin, evolution and function are discussed. The information presented in this review enables two main conclusions to be drawn. The first is that changes in the homeotic control of floral organ identity may have had a profound impact on floral structure in several disparate lineages in the family. This is most obvious in Fenerivia, in which a centrifugal shift of floral organ identity has occurred, and in Dasymaschalon, in which a reverse (centripetal) shift has occurred. Other genera that have gained or lost entire perianth whorls are likely to have undergone similar homeotic changes. Attention is also drawn to the extensive functional convergence in Annonaceae flowers, with widespread homoplasy in many characters that have previously been emphasized in higher‐level classifications.     

Pattern selection in a cooperative biochemical in vitro amplification system: the role of parasites

Previously, numerical simulations have shown that evolving systems can be stabilized against emerging parasites by pattern formation in spatially extended flow reactors. Hence, it can be argued that pattern formation is a prerequisite for any experimental investigation of the biochemical evolution of cooperative function. Here, we study a model of an experimental biochemical system for the cooperative in vitro amplification of DNA strands and show that emerging parasites can induce a complex pattern formation even when no pattern formation occurs without parasites. In an adiabatic approximation where the cooperative amplification reaction is assumed to adapt fast to slowly emerging parasites, the parasite concentration itself acts as a Steuer parameter for the selection of various complex patterns. Without such an adiabatic approximation only transient patterns emerge. As any species can grow for very low concentrations, the parasite is able to infect the entire reactor and the system is finally diluted out. In the experimental biochemical system, however, the species are individual molecules and the growth of spatially separated, non-infected regions becomes feasible. Hence a cutoff threshold for the minimal concentration is applied. In these simulations the otherwise lethal infection by parasites induces the formation of spatiotemporal spirals, and this spatial structure help the host and parasitoid species to survive together. These theoretical results describe an inherent property of cooperative reactions and have an important impact on experimental investigations on the molecular evolution and complex function in spatially extended reactors. Since the formation of the complex pattern is restricted either to a rather large cutoff value or a special choice of the kinetic parameters, we, however, conclude that the persistence of evolving cooperative amplification is not possible in a simple reaction-diffusion reactor. Experimental set-ups with patchy environments, e.g. biomolecular amplification in coupled microstructured flow chambers or in microemulsion, are eligible candidates for the observation of such a self-organized pattern selection.     

Regular patterns for proteome-wide distribution of protein abundance across species

A proteome of the bio-entity, including cell, tissue, organ, and organism, consists of proteins of diverse abundance. The principle that determines the abundance of different proteins in a proteome is of fundamental significance for an understanding of the building blocks of the bio-entity. Here, we report three regular patterns in the proteome-wide distribution of protein abundance across species such as human, mouse, fly, worm, yeast, and bacteria: in most cases, protein abundance is positively correlated with the protein's origination time or sequence conservation during evolution; it is negatively correlated with the protein's domain number and positively correlated with domain coverage in protein structure, and the correlations became stronger during the course of evolution; protein abundance can be further stratified by the function of the protein, whereby proteins that act on material conversion and transportation (mass category) are more abundant than those that act on information modulation (information category). Thus, protein abundance is intrinsically related to the protein's inherent characters of evolution, structure, and function.