Evolution of catalytic function

NASA Technical Reports Server (NTRS)

Joyce, G. F.

1993-01-01

An RNA-based evolution system was constructed in the laboratory and used to develop RNA enzymes with novel catalytic function. By controlling the nature of the catalytic task that the molecules must perform in order to survive, it is possible to direct the evolving population toward the expression of some desired catalytic behavior. More recently, this system has been coupled to an in vitro translation procedure, raising the possibility of evolving protein enzymes in the laboratory to produce novel proteins with desired catalytic properties. The aim of this line of research is to reduce darwinian evolution, the fundamental process of biology, to a laboratory procedure that can be made to operate in the service of organic synthesis.

Language evolution in the laboratory.

PubMed

Scott-Phillips, Thomas C; Kirby, Simon

2010-09-01

The historical origins of natural language cannot be observed directly. We can, however, study systems that support language and we can also develop models that explore the plausibility of different hypotheses about how language emerged. More recently, evolutionary linguists have begun to conduct language evolution experiments in the laboratory, where the emergence of new languages used by human participants can be observed directly. This enables researchers to study both the cognitive capacities necessary for language and the ways in which languages themselves emerge. One theme that runs through this work is how individual-level behaviours result in population-level linguistic phenomena. A central challenge for the future will be to explore how different forms of information transmission affect this process. 2010 Elsevier Ltd. All rights reserved.

Expanding the metabolic engineering toolbox with directed evolution.

PubMed

Abatemarco, Joseph; Hill, Andrew; Alper, Hal S

2013-12-01

Cellular systems can be engineered into factories that produce high-value chemicals from renewable feedstock. Such an approach requires an expanded toolbox for metabolic engineering. Recently, protein engineering and directed evolution strategies have started to play a growing and critical role within metabolic engineering. This review focuses on the various ways in which directed evolution can be applied in conjunction with metabolic engineering to improve product yields. Specifically, we discuss the application of directed evolution on both catalytic and non-catalytic traits of enzymes, on regulatory elements, and on whole genomes in a metabolic engineering context. We demonstrate how the goals of metabolic pathway engineering can be achieved in part through evolving cellular parts as opposed to traditional approaches that rely on gene overexpression and deletion. Finally, we discuss the current limitations in screening technology that hinder the full implementation of a metabolic pathway-directed evolution approach. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biophysical models of protein evolution: Understanding the patterns of evolutionary sequence divergence

PubMed Central

Echave, Julian; Wilke, Claus O.

2018-01-01

For decades, rates of protein evolution have been interpreted in terms of the vague concept of “functional importance”. Slowly evolving proteins or sites within proteins were assumed to be more functionally important and thus subject to stronger selection pressure. More recently, biophysical models of protein evolution, which combine evolutionary theory with protein biophysics, have completely revolutionized our view of the forces that shape sequence divergence. Slowly evolving proteins have been found to evolve slowly because of selection against toxic misfolding and misinteractions, linking their rate of evolution primarily to their abundance. Similarly, most slowly evolving sites in proteins are not directly involved in function, but mutating them has large impacts on protein structure and stability. Here, we review the studies of the emergent field of biophysical protein evolution that have shaped our current understanding of sequence divergence patterns. We also propose future research directions to develop this nascent field. PMID:28301766

Directed evolution: tailoring biocatalysts for industrial applications.

PubMed

Kumar, Ashwani; Singh, Suren

2013-12-01

Current challenges and promises of white biotechnology encourage protein engineers to use a directed evolution approach to generate novel and useful biocatalysts for various sets of applications. Different methods of enzyme engineering have been used in the past in an attempt to produce enzymes with improved functions and properties. Recent advancement in the field of random mutagenesis, screening, selection and computational design increased the versatility and the rapid development of enzymes under strong selection pressure with directed evolution experiments. Techniques of directed evolution improve enzymes fitness without understanding them in great detail and clearly demonstrate its future role in adapting enzymes for use in industry. Despite significant advances to date regarding biocatalyst improvement, there still remains a need to improve mutagenesis strategies and development of easy screening and selection tools without significant human intervention. This review covers fundamental and major development of directed evolution techniques, and highlights the advances in mutagenesis, screening and selection methods with examples of enzymes developed by using these approaches. Several commonly used methods for creating molecular diversity with their advantages and disadvantages including some recently used strategies are also discussed.

Directional Darwinian Selection in proteins.

PubMed

McClellan, David A

2013-01-01

Molecular evolution is a very active field of research, with several complementary approaches, including dN/dS, HON90, MM01, and others. Each has documented strengths and weaknesses, and no one approach provides a clear picture of how natural selection works at the molecular level. The purpose of this work is to present a simple new method that uses quantitative amino acid properties to identify and characterize directional selection in proteins. Inferred amino acid replacements are viewed through the prism of a single physicochemical property to determine the amount and direction of change caused by each replacement. This allows the calculation of the probability that the mean change in the single property associated with the amino acid replacements is equal to zero (H0: μ = 0; i.e., no net change) using a simple two-tailed t-test. Example data from calanoid and cyclopoid copepod cytochrome oxidase subunit I sequence pairs are presented to demonstrate how directional selection may be linked to major shifts in adaptive zones, and that convergent evolution at the whole organism level may be the result of convergent protein adaptations. Rather than replace previous methods, this new method further complements existing methods to provide a holistic glimpse of how natural selection shapes protein structure and function over evolutionary time.

Computational Protein Engineering: Bridging the Gap between Rational Design and Laboratory Evolution

PubMed Central

Barrozo, Alexandre; Borstnar, Rok; Marloie, Gaël; Kamerlin, Shina Caroline Lynn

2012-01-01

Enzymes are tremendously proficient catalysts, which can be used as extracellular catalysts for a whole host of processes, from chemical synthesis to the generation of novel biofuels. For them to be more amenable to the needs of biotechnology, however, it is often necessary to be able to manipulate their physico-chemical properties in an efficient and streamlined manner, and, ideally, to be able to train them to catalyze completely new reactions. Recent years have seen an explosion of interest in different approaches to achieve this, both in the laboratory, and in silico. There remains, however, a gap between current approaches to computational enzyme design, which have primarily focused on the early stages of the design process, and laboratory evolution, which is an extremely powerful tool for enzyme redesign, but will always be limited by the vastness of sequence space combined with the low frequency for desirable mutations. This review discusses different approaches towards computational enzyme design and demonstrates how combining newly developed screening approaches that can rapidly predict potential mutation “hotspots” with approaches that can quantitatively and reliably dissect the catalytic step can bridge the gap that currently exists between computational enzyme design and laboratory evolution studies. PMID:23202907

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat.

PubMed

Jeong, Haeyoung; Lee, Sang J; Kim, Pil

2016-09-20

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Mapping the Geometric Evolution of Protein Folding Motor.

PubMed

Jerath, Gaurav; Hazam, Prakash Kishore; Shekhar, Shashi; Ramakrishnan, Vibin

2016-01-01

Polypeptide chain has an invariant main-chain and a variant side-chain sequence. How the side-chain sequence determines fold in terms of its chemical constitution has been scrutinized extensively and verified periodically. However, a focussed investigation on the directive effect of side-chain geometry may provide important insights supplementing existing algorithms in mapping the geometrical evolution of protein chains and its structural preferences. Geometrically, folding of protein structure may be envisaged as the evolution of its geometric variables: ϕ, and ψ dihedral angles of polypeptide main-chain directed by χ1, and χ2 of side chain. In this work, protein molecule is metaphorically modelled as a machine with 4 rotors ϕ, ψ, χ1 and χ2, with its evolution to the functional fold is directed by combinations of its rotor directions. We observe that differential rotor motions lead to different secondary structure formations and the combinatorial pattern is unique and consistent for particular secondary structure type. Further, we found that combination of rotor geometries of each amino acid is unique which partly explains how different amino acid sequence combinations have unique structural evolution and functional adaptation. Quantification of these amino acid rotor preferences, resulted in the generation of 3 substitution matrices, which later on plugged in the BLAST tool, for evaluating their efficiency in aligning sequences. We have employed BLOSUM62 and PAM30 as standard for primary evaluation. Generation of substitution matrices is a logical extension of the conceptual framework we attempted to build during the development of this work. Optimization of matrices following the conventional routines and possible application with biologically relevant data sets are beyond the scope of this manuscript, though it is a part of the larger project design.

Convergent evolution and mimicry of protein linear motifs in host-pathogen interactions.

PubMed

Chemes, Lucía Beatriz; de Prat-Gay, Gonzalo; Sánchez, Ignacio Enrique

2015-06-01

Pathogen linear motif mimics are highly evolvable elements that facilitate rewiring of host protein interaction networks. Host linear motifs and pathogen mimics differ in sequence, leading to thermodynamic and structural differences in the resulting protein-protein interactions. Moreover, the functional output of a mimic depends on the motif and domain repertoire of the pathogen protein. Regulatory evolution mediated by linear motifs can be understood by measuring evolutionary rates, quantifying positive and negative selection and performing phylogenetic reconstructions of linear motif natural history. Convergent evolution of linear motif mimics is widespread among unrelated proteins from viral, prokaryotic and eukaryotic pathogens and can also take place within individual protein phylogenies. Statistics, biochemistry and laboratory models of infection link pathogen linear motifs to phenotypic traits such as tropism, virulence and oncogenicity. In vitro evolution experiments and analysis of natural sequences suggest that changes in linear motif composition underlie pathogen adaptation to a changing environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Continuous directed evolution of aminoacyl-tRNA synthetases

PubMed Central

Bryson, David I.; Fan, Chenguang; Guo, Li-Tao; Miller, Corwin; Söll, Dieter; Liu, David R.

2017-01-01

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of non-canonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing non-canonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity. PMID:29035361

Directed Chemical Evolution with an Outsized Genetic Code

PubMed Central

Krusemark, Casey J.; Tilmans, Nicolas P.; Brown, Patrick O.; Harbury, Pehr B.

2016-01-01

The first demonstration that macromolecules could be evolved in a test tube was reported twenty-five years ago. That breakthrough meant that billions of years of chance discovery and refinement could be compressed into a few weeks, and provided a powerful tool that now dominates all aspects of protein engineering. A challenge has been to extend this scientific advance into synthetic chemical space: to enable the directed evolution of abiotic molecules. The problem has been tackled in many ways. These include expanding the natural genetic code to include unnatural amino acids, engineering polyketide and polypeptide synthases to produce novel products, and tagging combinatorial chemistry libraries with DNA. Importantly, there is still no small-molecule analog of directed protein evolution, i.e. a substantiated approach for optimizing complex (≥ 10^9 diversity) populations of synthetic small molecules over successive generations. We present a key advance towards this goal: a tool for genetically-programmed synthesis of small-molecule libraries from large chemical alphabets. The approach accommodates alphabets that are one to two orders of magnitude larger than any in Nature, and facilitates evolution within the chemical spaces they create. This is critical for small molecules, which are built up from numerous and highly varied chemical fragments. We report a proof-of-concept chemical evolution experiment utilizing an outsized genetic code, and demonstrate that fitness traits can be passed from an initial small-molecule population through to the great-grandchildren of that population. The results establish the practical feasibility of engineering synthetic small molecules through accelerated evolution. PMID:27508294

Directed evolution of artificial metalloenzymes for in vivo metathesis

NASA Astrophysics Data System (ADS)

Jeschek, Markus; Reuter, Raphael; Heinisch, Tillmann; Trindler, Christian; Klehr, Juliane; Panke, Sven; Ward, Thomas R.

2016-09-01

The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions. For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist: repurposing natural metalloenzymes for abiotic transformations; in silico metalloenzyme (re-)design; and incorporation of abiotic cofactors into proteins. The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein. This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction without equivalent in nature. Building on previous work on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in

Biophysics of protein evolution and evolutionary protein biophysics

PubMed Central

Sikosek, Tobias; Chan, Hue Sun

2014-01-01

The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence–structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by ‘hidden’ conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution. PMID:25165599

Adaptive laboratory evolution -- principles and applications for biotechnology.

PubMed

Dragosits, Martin; Mattanovich, Diethard

2013-07-01

Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations. However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design. Over the last two decades important insights into nutrient and stress metabolism of relevant model species were acquired, whereas some other aspects such as niche-specific differences of non-conventional cell factories are not completely understood. Altogether the current status and its future perspectives highlight the importance and potential of adaptive laboratory evolution as approach in biotechnological engineering.

Evolution of proteins.

NASA Technical Reports Server (NTRS)

Dayhoff, M. O.

1971-01-01

The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

Two fundamental questions about protein evolution.

PubMed

Penny, David; Zhong, Bojian

2015-12-01

Two basic questions are considered that approach protein evolution from different directions; the problems arising from using Markov models for the deeper divergences, and then the origin of proteins themselves. The real problem for the first question (going backwards in time) is that at deeper phylogenies the Markov models of sequence evolution must lose information exponentially at deeper divergences, and several testable methods are suggested that should help resolve these deeper divergences. For the second question (coming forwards in time) a problem is that most models for the origin of protein synthesis do not give a role for the very earliest stages of the process. From our knowledge of the importance of replication accuracy in limiting the length of a coding molecule, a testable hypothesis is proposed. The length of the code, the code itself, and tRNAs would all have prior roles in increasing the accuracy of RNA replication; thus proteins would have been formed only after the tRNAs and the length of the triplet code are already formed. Both questions lead to testable predictions. Copyright © 2014 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Fluorescence quantum yield measurements of fluorescent proteins: a laboratory experiment for a biochemistry or molecular biophysics laboratory course.

PubMed

Wall, Kathryn P; Dillon, Rebecca; Knowles, Michelle K

2015-01-01

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper-level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum. © 2014 The International Union of Biochemistry and Molecular Biology.

Adaptive laboratory evolution – principles and applications for biotechnology

PubMed Central

2013-01-01

Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations. However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design. Over the last two decades important insights into nutrient and stress metabolism of relevant model species were acquired, whereas some other aspects such as niche-specific differences of non-conventional cell factories are not completely understood. Altogether the current status and its future perspectives highlight the importance and potential of adaptive laboratory evolution as approach in biotechnological engineering. PMID:23815749

Metal-directed design of supramolecular protein assemblies

PubMed Central

Bailey, Jake B.; Subramanian, Rohit H.; Churchfield, Lewis A.

2016-01-01

Owing to their central roles in cellular signaling, construction, and biochemistry, protein-protein interactions (PPIs) and protein self-assembly have become a major focus of molecular design and synthetic biology. In order to circumvent the complexity of constructing extensive non-covalent interfaces, which are typically involved in natural PPIs and protein self-assembly, we have developed two design strategies, Metal-Directed Protein Self-Assembly (MDPSA) and Metal-Templated Interface Redesign (MeTIR). These strategies, inspired by both the proposed evolutionary roles of metals and their prevalence in natural PPIs, take advantage of the favorable properties of metal coordination (bonding strength, directionality, and reversibility) to guide protein self-assembly with minimal design and engineering. Using a small, monomeric protein (cytochrome cb562) as a model building block, we employed MDPSA and MeTIR to create a diverse array of functional supramolecular architectures which range from structurally tunable oligomers to metalloprotein complexes that can properly self-assemble in living cells into novel metalloenzymes. The design principles and strategies outlined herein should be readily applicable to other protein systems with the goal of creating new PPIs and protein assemblies with structures and functions not yet produced by natural evolution. PMID:27586336

Directed evolution of cell size in Escherichia coli.

PubMed

Yoshida, Mari; Tsuru, Saburo; Hirata, Naoko; Seno, Shigeto; Matsuda, Hideo; Ying, Bei-Wen; Yomo, Tetsuya

2014-12-17

In bacteria, cell size affects chromosome replication, the assembly of division machinery, cell wall synthesis, membrane synthesis and ultimately growth rate. In addition, cell size can also be a target for Darwinian evolution for protection from predators. This strong coupling of cell size and growth, however, could lead to the introduction of growth defects after size evolution. An important question remains: can bacterial cell size change and/or evolve without imposing a growth burden? The directed evolution of particular cell sizes, without a growth burden, was tested with a laboratory Escherichia coli strain. Cells of defined size ranges were collected by a cell sorter and were subsequently cultured. This selection-propagation cycle was repeated, and significant changes in cell size were detected within 400 generations. In addition, the width of the size distribution was altered. The changes in cell size were unaccompanied by a growth burden. Whole genome sequencing revealed that only a few mutations in genes related to membrane synthesis conferred the size evolution. In conclusion, bacterial cell size could evolve, through a few mutations, without growth reduction. The size evolution without growth reduction suggests a rapid evolutionary change to diverse cell sizes in bacterial survival strategies.

A Model of Substitution Trajectories in Sequence Space and Long-Term Protein Evolution

PubMed Central

Usmanova, Dinara R.; Ferretti, Luca; Povolotskaya, Inna S.; Vlasov, Peter K.; Kondrashov, Fyodor A.

2015-01-01

The nature of factors governing the tempo and mode of protein evolution is a fundamental issue in evolutionary biology. Specifically, whether or not interactions between different sites, or epistasis, are important in directing the course of evolution became one of the central questions. Several recent reports have scrutinized patterns of long-term protein evolution claiming them to be compatible only with an epistatic fitness landscape. However, these claims have not yet been substantiated with a formal model of protein evolution. Here, we formulate a simple covarion-like model of protein evolution focusing on the rate at which the fitness impact of amino acids at a site changes with time. We then apply the model to the data on convergent and divergent protein evolution to test whether or not the incorporation of epistatic interactions is necessary to explain the data. We find that convergent evolution cannot be explained without the incorporation of epistasis and the rate at which an amino acid state switches from being acceptable at a site to being deleterious is faster than the rate of amino acid substitution. Specifically, for proteins that have persisted in modern prokaryotic organisms since the last universal common ancestor for one amino acid substitution approximately ten amino acid states switch from being accessible to being deleterious, or vice versa. Thus, molecular evolution can only be perceived in the context of rapid turnover of which amino acids are available for evolution. PMID:25415964

Streamlining workflow and automation to accelerate laboratory scale protein production.

PubMed

Konczal, Jennifer; Gray, Christopher H

2017-05-01

Protein production facilities are often required to produce diverse arrays of proteins for demanding methodologies including crystallography, NMR, ITC and other reagent intensive techniques. It is common for these teams to find themselves a bottleneck in the pipeline of ambitious projects. This pressure to deliver has resulted in the evolution of many novel methods to increase capacity and throughput at all stages in the pipeline for generation of recombinant proteins. This review aims to describe current and emerging options to accelerate the success of protein production in Escherichia coli. We emphasize technologies that have been evaluated and implemented in our laboratory, including innovative molecular biology and expression vectors, small-scale expression screening strategies and the automation of parallel and multidimensional chromatography. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The role of protein dynamics in the evolution of new enzyme function.

PubMed

Campbell, Eleanor; Kaltenbach, Miriam; Correy, Galen J; Carr, Paul D; Porebski, Benjamin T; Livingstone, Emma K; Afriat-Jurnou, Livnat; Buckle, Ashley M; Weik, Martin; Hollfelder, Florian; Tokuriki, Nobuhiko; Jackson, Colin J

2016-11-01

Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.

Evolution of cyclohexadienyl dehydratase from an ancestral solute-binding protein.

PubMed

Clifton, Ben E; Kaczmarski, Joe A; Carr, Paul D; Gerth, Monica L; Tokuriki, Nobuhiko; Jackson, Colin J

2018-04-23

The emergence of enzymes through the neofunctionalization of noncatalytic proteins is ultimately responsible for the extraordinary range of biological catalysts observed in nature. Although the evolution of some enzymes from binding proteins can be inferred by homology, we have a limited understanding of the nature of the biochemical and biophysical adaptations along these evolutionary trajectories and the sequence in which they occurred. Here we reconstructed and characterized evolutionary intermediate states linking an ancestral solute-binding protein to the extant enzyme cyclohexadienyl dehydratase. We show how the intrinsic reactivity of a desolvated general acid was harnessed by a series of mutations radiating from the active site, which optimized enzyme-substrate complementarity and transition-state stabilization and minimized sampling of noncatalytic conformations. Our work reveals the molecular evolutionary processes that underlie the emergence of enzymes de novo, which are notably mirrored by recent examples of computational enzyme design and directed evolution.

Evolution of reproductive proteins from animals and plants.

PubMed

Clark, Nathaniel L; Aagaard, Jan E; Swanson, Willie J

2006-01-01

Sexual reproduction is a fundamental biological process common among eukaryotes. Because of the significance of reproductive proteins to fitness, the diversity and rapid divergence of proteins acting at many stages of reproduction is surprising and suggests a role of adaptive diversification in reproductive protein evolution. Here we review the evolution of reproductive proteins acting at different stages of reproduction among animals and plants, emphasizing common patterns. Although we are just beginning to understand these patterns, by making comparisons among stages of reproduction for diverse organisms we can begin to understand the selective forces driving reproductive protein diversity and the functional consequences of reproductive protein evolution.

Understanding protein evolution: from protein physics to Darwinian selection.

PubMed

Zeldovich, Konstantin B; Shakhnovich, Eugene I

2008-01-01

Efforts in whole-genome sequencing and structural proteomics start to provide a global view of the protein universe, the set of existing protein structures and sequences. However, approaches based on the selection of individual sequences have not been entirely successful at the quantitative description of the distribution of structures and sequences in the protein universe because evolutionary pressure acts on the entire organism, rather than on a particular molecule. In parallel to this line of study, studies in population genetics and phenomenological molecular evolution established a mathematical framework to describe the changes in genome sequences in populations of organisms over time. Here, we review both microscopic (physics-based) and macroscopic (organism-level) models of protein-sequence evolution and demonstrate that bridging the two scales provides the most complete description of the protein universe starting from clearly defined, testable, and physiologically relevant assumptions.

Intermediate filament protein evolution and protists.

PubMed

Preisner, Harald; Habicht, Jörn; Garg, Sriram G; Gould, Sven B

2018-03-23

Metazoans evolved from a single protist lineage. While all eukaryotes share a conserved actin and tubulin-based cytoskeleton, it is commonly perceived that intermediate filaments (IFs), including lamin, vimentin or keratin among many others, are restricted to metazoans. Actin and tubulin proteins are conserved enough to be detectable across all eukaryotic genomes using standard phylogenetic methods, but IF proteins, in contrast, are notoriously difficult to identify by such means. Since the 1950s, dozens of cytoskeletal proteins in protists have been identified that seemingly do not belong to any of the IF families described for metazoans, yet, from a structural and functional perspective fit criteria that define metazoan IF proteins. Here, we briefly review IF protein discovery in metazoans and the implications this had for the definition of this protein family. We argue that the many cytoskeletal and filament-forming proteins of protists should be incorporated into a more comprehensive picture of IF evolution by aligning it with the recent identification of lamins across the phylogenetic diversity of eukaryotic supergroups. This then brings forth the question of how the diversity of IF proteins has unfolded. The evolution of IF proteins likely represents an example of convergent evolution, which, in combination with the speed with which these cytoskeletal proteins are evolving, generated their current diversity. IF proteins did not first emerge in metazoa, but in protists. Only the emergence of cytosolic IF proteins that appear to stem from a nuclear lamin is unique to animals and coincided with the emergence of true animal multicellularity. © 2018 Wiley Periodicals, Inc.

Compartmentalized partnered replication for the directed evolution of genetic parts and circuits.

PubMed

Abil, Zhanar; Ellefson, Jared W; Gollihar, Jimmy D; Watkins, Ella; Ellington, Andrew D

2017-12-01

Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype-genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection. Here, we present a step-by-step guideline for the procedure by providing examples of (i) selection of T7 RNA polymerases that recognize orthogonal promoters and (ii) selection of tRNA for enhanced amber codon suppression. A single round of CPR should take ∼3-5 d, whereas a whole directed evolution can be performed in 3-10 rounds, depending on selection efficiency.

Protein trans-splicing on an M13 bacteriophage: towards directed evolution of a semisynthetic split intein by phage display.

PubMed

Garbe, Daniel; Thiel, Ilka V; Mootz, Henning D

2010-10-01

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties. © 2010 European Peptide Society and John Wiley & Sons, Ltd.

Position Matters: Network Centrality Considerably Impacts Rates of Protein Evolution in the Human Protein-Protein Interaction Network.

PubMed

Alvarez-Ponce, David; Feyertag, Felix; Chakraborty, Sandip

2017-06-01

The proteins of any organism evolve at disparate rates. A long list of factors affecting rates of protein evolution have been identified. However, the relative importance of each factor in determining rates of protein evolution remains unresolved. The prevailing view is that evolutionary rates are dominantly determined by gene expression, and that other factors such as network centrality have only a marginal effect, if any. However, this view is largely based on analyses in yeasts, and accurately measuring the importance of the determinants of rates of protein evolution is complicated by the fact that the different factors are often correlated with each other, and by the relatively poor quality of available functional genomics data sets. Here, we use correlation, partial correlation and principal component regression analyses to measure the contributions of several factors to the variability of the rates of evolution of human proteins. For this purpose, we analyzed the entire human protein-protein interaction data set and the human signal transduction network-a network data set of exceptionally high quality, obtained by manual curation, which is expected to be virtually free from false positives. In contrast with the prevailing view, we observe that network centrality (measured as the number of physical and nonphysical interactions, betweenness, and closeness) has a considerable impact on rates of protein evolution. Surprisingly, the impact of centrality on rates of protein evolution seems to be comparable, or even superior according to some analyses, to that of gene expression. Our observations seem to be independent of potentially confounding factors and from the limitations (biases and errors) of interactomic data sets. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The interface of protein structure, protein biophysics, and molecular evolution

PubMed Central

Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon

2012-01-01

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

Evolution of Protein Domain Repeats in Metazoa

PubMed Central

Schüler, Andreas; Bornberg-Bauer, Erich

2016-01-01

Repeats are ubiquitous elements of proteins and they play important roles for cellular function and during evolution. Repeats are, however, also notoriously difficult to capture computationally and large scale studies so far had difficulties in linking genetic causes, structural properties and evolutionary trajectories of protein repeats. Here we apply recently developed methods for repeat detection and analysis to a large dataset comprising over hundred metazoan genomes. We find that repeats in larger protein families experience generally very few insertions or deletions (indels) of repeat units but there is also a significant fraction of noteworthy volatile outliers with very high indel rates. Analysis of structural data indicates that repeats with an open structure and independently folding units are more volatile and more likely to be intrinsically disordered. Such disordered repeats are also significantly enriched in sites with a high functional potential such as linear motifs. Furthermore, the most volatile repeats have a high sequence similarity between their units. Since many volatile repeats also show signs of recombination, we conclude they are often shaped by concerted evolution. Intriguingly, many of these conserved yet volatile repeats are involved in host-pathogen interactions where they might foster fast but subtle adaptation in biological arms races. Key Words: protein evolution, domain rearrangements, protein repeats, concerted evolution. PMID:27671125

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template.

PubMed

Gouran, Hossein; Chakraborty, Sandeep; Rao, Basuthkar J; Asgeirsson, Bjarni; Dandekar, Abhaya

2014-01-01

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction.

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template

PubMed Central

Rao, Basuthkar J.; Asgeirsson, Bjarni; Dandekar, Abhaya

2014-01-01

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction. PMID:25717364

Sandia National Laboratories: Research: Laboratory Directed Research &

Science.gov Websites

; Technology Defense Systems & Assessments About Defense Systems & Assessments Program Areas Robotics R&D 100 Awards Laboratory Directed Research & Development Technology Deployment Centers Audit Sandia's Economic Impact Licensing & Technology Transfer Browse Technology Portfolios

Determinants of the rate of protein sequence evolution

PubMed Central

Zhang, Jianzhi; Yang, Jian-Rong

2015-01-01

The rate and mechanism of protein sequence evolution have been central questions in evolutionary biology since the 1960s. Although the rate of protein sequence evolution depends primarily on the level of functional constraint, exactly what constitutes functional constraint has remained unclear. The increasing availability of genomic data has allowed for much needed empirical examinations on the nature of functional constraint. These studies found that the evolutionary rate of a protein is predominantly influenced by its expression level rather than functional importance. A combination of theoretical and empirical analyses have identified multiple mechanisms behind these observations and demonstrated a prominent role that selection against errors in molecular and cellular processes plays in protein evolution. PMID:26055156

A Generative Angular Model of Protein Structure Evolution

PubMed Central

Golden, Michael; García-Portugués, Eduardo; Sørensen, Michael; Mardia, Kanti V.; Hamelryck, Thomas; Hein, Jotun

2017-01-01

Abstract Recently described stochastic models of protein evolution have demonstrated that the inclusion of structural information in addition to amino acid sequences leads to a more reliable estimation of evolutionary parameters. We present a generative, evolutionary model of protein structure and sequence that is valid on a local length scale. The model concerns the local dependencies between sequence and structure evolution in a pair of homologous proteins. The evolutionary trajectory between the two structures in the protein pair is treated as a random walk in dihedral angle space, which is modeled using a novel angular diffusion process on the two-dimensional torus. Coupling sequence and structure evolution in our model allows for modeling both “smooth” conformational changes and “catastrophic” conformational jumps, conditioned on the amino acid changes. The model has interpretable parameters and is comparatively more realistic than previous stochastic models, providing new insights into the relationship between sequence and structure evolution. For example, using the trained model we were able to identify an apparent sequence–structure evolutionary motif present in a large number of homologous protein pairs. The generative nature of our model enables us to evaluate its validity and its ability to simulate aspects of protein evolution conditioned on an amino acid sequence, a related amino acid sequence, a related structure or any combination thereof. PMID:28453724

Simulation of gene evolution under directional mutational pressure

NASA Astrophysics Data System (ADS)

Dudkiewicz, Małgorzata; Mackiewicz, Paweł; Kowalczuk, Maria; Mackiewicz, Dorota; Nowicka, Aleksandra; Polak, Natalia; Smolarczyk, Kamila; Banaszak, Joanna; R. Dudek, Mirosław; Cebrat, Stanisław

2004-05-01

The two main mechanisms generating the genetic diversity, mutation and recombination, have random character but they are biased which has an effect on the generation of asymmetry in the bacterial chromosome structure and in the protein coding sequences. Thus, like in a case of two chiral molecules-the two possible orientations of a gene in relation to the topology of a chromosome are not equivalent. Assuming that the sequence of a gene may oscillate only between certain limits of its structural composition means that the gene could be forced out of these limits by the directional mutation pressure, in the course of evolution. The probability of the event depends on the time the gene stays under the same mutation pressure. Inversion of the gene changes the directional mutational pressure to the reciprocal one and hence it changes the distance of the gene to its lower and upper bound of the structural tolerance. Using Monte Carlo methods we were able to simulate the evolution of genes under experimentally found mutational pressure, assuming simple mechanisms of selection. We found that the mutation and recombination should work in accordance to lower their negative effects on the function of the products of coding sequences.

Origin and evolution of chromosomal sperm proteins.

PubMed

Eirín-López, José M; Ausió, Juan

2009-10-01

In the eukaryotic cell, DNA compaction is achieved through its interaction with histones, constituting a nucleoprotein complex called chromatin. During metazoan evolution, the different structural and functional constraints imposed on the somatic and germinal cell lines led to a unique process of specialization of the sperm nuclear basic proteins (SNBPs) associated with chromatin in male germ cells. SNBPs encompass a heterogeneous group of proteins which, since their discovery in the nineteenth century, have been studied extensively in different organisms. However, the origin and controversial mechanisms driving the evolution of this group of proteins has only recently started to be understood. Here, we analyze in detail the histone hypothesis for the vertical parallel evolution of SNBPs, involving a "vertical" transition from a histone to a protamine-like and finally protamine types (H --> PL --> P), the last one of which is present in the sperm of organisms at the uppermost tips of the phylogenetic tree. In particular, the common ancestry shared by the protamine-like (PL)- and protamine (P)-types with histone H1 is discussed within the context of the diverse structural and functional constraints acting upon these proteins during bilaterian evolution.

Successive gain of insulator proteins in arthropod evolution.

PubMed

Heger, Peter; George, Rebecca; Wiehe, Thomas

2013-10-01

Alteration of regulatory DNA elements or their binding proteins may have drastic consequences for morphological evolution. Chromatin insulators are one example of such proteins and play a fundamental role in organizing gene expression. While a single insulator protein, CTCF (CCCTC-binding factor), is known in vertebrates, Drosophila melanogaster utilizes six additional factors. We studied the evolution of these proteins and show here that-in contrast to the bilaterian-wide distribution of CTCF-all other D. melanogaster insulators are restricted to arthropods. The full set is present exclusively in the genus Drosophila whereas only two insulators, Su(Hw) and CTCF, existed at the base of the arthropod clade and all additional factors have been acquired successively at later stages. Secondary loss of factors in some lineages further led to the presence of different insulator subsets in arthropods. Thus, the evolution of insulator proteins within arthropods is an ongoing and dynamic process that reshapes and supplements the ancient CTCF-based system common to bilaterians. Expansion of insulator systems may therefore be a general strategy to increase an organism's gene regulatory repertoire and its potential for morphological plasticity. © 2013 The Authors. Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.

Global Clusters as Laboratories for Stellar Evolution

NASA Technical Reports Server (NTRS)

Catelan, Marcio; Valcarce, Aldo A. R.; Sweigart, Allen V.

2010-01-01

Globular clusters have long been considered the closest approximation to a physicist's laboratory in astrophysics, and as such a near-ideal laboratory for (low-mass) stellar evolution, However, recent observations have cast a shadow on this long-standing paradigm, suggesting the presence of multiple populations with widely different abundance patterns, and - crucially - with widely different helium abundances as welL In this review we discuss which features of the Hertzsprung-Russell diagram may be used as helium abundance indicators, and present an overview of available constraints on the helium abundance in globular clusters,

Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum.

PubMed

Mahr, Regina; Gätgens, Cornelia; Gätgens, Jochem; Polen, Tino; Kalinowski, Jörn; Frunzke, Julia

2015-11-01

Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The L-valine producer Corynebacterium glutamicum ΔaceE was equipped with an L-valine-responsive sensor based on the transcriptional regulator Lrp of C. glutamicum. Evolved strains featured a significantly higher growth rate, increased L-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188*) in the gene ureD (urease accessory protein), which was shown to increase L-valine production by up to 100%. Furthermore, decreased L-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Music Evolution in the Laboratory: Cultural Transmission Meets Neurophysiology.

PubMed

Lumaca, Massimo; Ravignani, Andrea; Baggio, Giosuè

2018-01-01

In recent years, there has been renewed interest in the biological and cultural evolution of music, and specifically in the role played by perceptual and cognitive factors in shaping core features of musical systems, such as melody, harmony, and rhythm. One proposal originates in the language sciences. It holds that aspects of musical systems evolve by adapting gradually, in the course of successive generations, to the structural and functional characteristics of the sensory and memory systems of learners and "users" of music. This hypothesis has found initial support in laboratory experiments on music transmission. In this article, we first review some of the most important theoretical and empirical contributions to the field of music evolution. Next, we identify a major current limitation of these studies, i.e., the lack of direct neural support for the hypothesis of cognitive adaptation. Finally, we discuss a recent experiment in which this issue was addressed by using event-related potentials (ERPs). We suggest that the introduction of neurophysiology in cultural transmission research may provide novel insights on the micro-evolutionary origins of forms of variation observed in cultural systems.

Music Evolution in the Laboratory: Cultural Transmission Meets Neurophysiology

PubMed Central

Lumaca, Massimo; Ravignani, Andrea; Baggio, Giosuè

2018-01-01

In recent years, there has been renewed interest in the biological and cultural evolution of music, and specifically in the role played by perceptual and cognitive factors in shaping core features of musical systems, such as melody, harmony, and rhythm. One proposal originates in the language sciences. It holds that aspects of musical systems evolve by adapting gradually, in the course of successive generations, to the structural and functional characteristics of the sensory and memory systems of learners and “users” of music. This hypothesis has found initial support in laboratory experiments on music transmission. In this article, we first review some of the most important theoretical and empirical contributions to the field of music evolution. Next, we identify a major current limitation of these studies, i.e., the lack of direct neural support for the hypothesis of cognitive adaptation. Finally, we discuss a recent experiment in which this issue was addressed by using event-related potentials (ERPs). We suggest that the introduction of neurophysiology in cultural transmission research may provide novel insights on the micro-evolutionary origins of forms of variation observed in cultural systems. PMID:29713263

Protein Laboratories in Single Location | Poster

Cancer.gov

By Andrew Stephen, Timothy Veenstra, and Gordon Whiteley, Guest Writers, and Ken Michaels, Staff Writer The Laboratory of Proteomics and Analytical Technologies (LPAT), Antibody Characterization Laboratory (ACL), and Protein Chemistry Laboratory (PCL), previously located on different floors or in different buildings, are now together on the first floor of C wing in the ATRF.

Evolution of Protein Synthesis from an RNA World

PubMed Central

Noller, Harry F.

2012-01-01

SUMMARY Because of the molecular complexity of the ribosome and protein synthesis, it is a challenge to imagine how translation could have evolved from a primitive RNA World. Two specific suggestions are made here to help to address this, involving separate evolution of the peptidyl transferase and decoding functions. First, it is proposed that translation originally arose not to synthesize functional proteins, but to provide simple (perhaps random) peptides that bound to RNA, increasing its available structure space, and therefore its functional capabilities. Second, it is proposed that the decoding site of the ribosome evolved from a mechanism for duplication of RNA. This process involved homodimeric “duplicator RNAs,” resembling the anticodon arms of tRNAs, which directed ligation of trinucleotides in response to an RNA template. PMID:20610545

Incorporating information on predicted solvent accessibility to the co-evolution-based study of protein interactions.

PubMed

Ochoa, David; García-Gutiérrez, Ponciano; Juan, David; Valencia, Alfonso; Pazos, Florencio

2013-01-27

A widespread family of methods for studying and predicting protein interactions using sequence information is based on co-evolution, quantified as similarity of phylogenetic trees. Part of the co-evolution observed between interacting proteins could be due to co-adaptation caused by inter-protein contacts. In this case, the co-evolution is expected to be more evident when evaluated on the surface of the proteins or the internal layers close to it. In this work we study the effect of incorporating information on predicted solvent accessibility to three methods for predicting protein interactions based on similarity of phylogenetic trees. We evaluate the performance of these methods in predicting different types of protein associations when trees based on positions with different characteristics of predicted accessibility are used as input. We found that predicted accessibility improves the results of two recent versions of the mirrortree methodology in predicting direct binary physical interactions, while it neither improves these methods, nor the original mirrortree method, in predicting other types of interactions. That improvement comes at no cost in terms of applicability since accessibility can be predicted for any sequence. We also found that predictions of protein-protein interactions are improved when multiple sequence alignments with a richer representation of sequences (including paralogs) are incorporated in the accessibility prediction.

Laboratory directed research and development program, FY 1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1997-02-01

The Ernest Orlando Lawrence Berkeley National Laboratory (Berkeley Lab) Laboratory Directed Research and Development Program FY 1996 report is compiled from annual reports submitted by principal investigators following the close of the fiscal year. This report describes the projects supported and summarizes their accomplishments. It constitutes a part of the Laboratory Directed Research and Development (LDRD) program planning and documentation process that includes an annual planning cycle, projection selection, implementation, and review. The Berkeley Lab LDRD program is a critical tool for directing the Laboratory`s forefront scientific research capabilities toward vital, excellent, and emerging scientific challenges. The program provides themore » resources for Berkeley Lab scientists to make rapid and significant contributions to critical national science and technology problems. The LDRD program also advances the Laboratory`s core competencies, foundations, and scientific capability, and permits exploration of exciting new opportunities. Areas eligible for support include: (1) Work in forefront areas of science and technology that enrich Laboratory research and development capability; (2) Advanced study of new hypotheses, new experiments, and innovative approaches to develop new concepts or knowledge; (3) Experiments directed toward proof of principle for initial hypothesis testing or verification; and (4) Conception and preliminary technical analysis to explore possible instrumentation, experimental facilities, or new devices.« less

Exploring the evolution of protein function in Archaea.

PubMed

Goncearenco, Alexander; Berezovsky, Igor N

2012-05-30

Despite recent progress in studies of the evolution of protein function, the questions what were the first functional protein domains and what were their basic building blocks remain unresolved. Previously, we introduced the concept of elementary functional loops (EFLs), which are the functional units of enzymes that provide elementary reactions in biochemical transformations. They are presumably descendants of primordial catalytic peptides. We analyzed distant evolutionary connections between protein functions in Archaea based on the EFLs comprising them. We show examples of the involvement of EFLs in new functional domains, as well as reutilization of EFLs and functional domains in building multidomain structures and protein complexes. Our analysis of the archaeal superkingdom yields the dominating mechanisms in different periods of protein evolution, which resulted in several levels of the organization of biochemical function. First, functional domains emerged as combinations of prebiotic peptides with the very basic functions, such as nucleotide/phosphate and metal cofactor binding. Second, domain recombination brought to the evolutionary scene the multidomain proteins and complexes. Later, reutilization and de novo design of functional domains and elementary functional loops complemented evolution of protein function.

Evolution of Protein Lipograms: A Bioinformatics Problem

ERIC Educational Resources Information Center

White, Harold B., III; Dhurjati, Prasad

2006-01-01

A protein lacking one of the 20 common amino acids is a protein lipogram. This open-ended problem-based learning assignment deals with the evolution of proteins with biased amino acid composition. It has students query protein and metabolic databases to test the hypothesis that natural selection has reduced the frequency of each amino acid…

Dynamic Network-Based Relevance Score Reveals Essential Proteins and Functional Modules in Directed Differentiation

PubMed Central

Wu, Chia-Chou; Lin, Che

2015-01-01

The induction of stem cells toward a desired differentiation direction is required for the advancement of stem cell-based therapies. Despite successful demonstrations of the control of differentiation direction, the effective use of stem cell-based therapies suffers from a lack of systematic knowledge regarding the mechanisms underlying directed differentiation. Using dynamic modeling and the temporal microarray data of three differentiation stages, three dynamic protein-protein interaction networks were constructed. The interaction difference networks derived from the constructed networks systematically delineated the evolution of interaction variations and the underlying mechanisms. A proposed relevance score identified the essential components in the directed differentiation. Inspection of well-known proteins and functional modules in the directed differentiation showed the plausibility of the proposed relevance score, with the higher scores of several proteins and function modules indicating their essential roles in the directed differentiation. During the differentiation process, the proteins and functional modules with higher relevance scores also became more specific to the neuronal identity. Ultimately, the essential components revealed by the relevance scores may play a role in controlling the direction of differentiation. In addition, these components may serve as a starting point for understanding the systematic mechanisms of directed differentiation and for increasing the efficiency of stem cell-based therapies. PMID:25977693

Evolution of catalytic RNA in the laboratory

NASA Technical Reports Server (NTRS)

Joyce, Gerald F.

1992-01-01

We are interested in the biochemistry of existing RNA enzymes and in the development of RNA enzymes with novel catalytic function. The focal point of our research program has been the design and operation of a laboratory system for the controlled evolution of catalytic RNA. This system serves as working model of RNA-based life and can be used to explore the catalytic potential of RNA. Evolution requires the integration of three chemical processes: amplification, mutation, and selection. Amplification results in additional copies of the genetic material. Mutation operates at the level of genotype to introduce variability, this variability in turn being expressed as a range of phenotypes. Selection operates at the level of phenotype to reduce variability by excluding those individuals that do not conform to the prevailing fitness criteria. These three processes must be linked so that only the selected individuals are amplified, subject to mutational error, to produce a progeny distribution of mutant individuals. We devised techniques for the amplification, mutation, and selection of catalytic RNA, all of which can be performed rapidly in vitro within a single reaction vessel. We integrated these techniques in such a way that they can be performed iteratively and routinely. This allowed us to conduct evolution experiments in response to artificially-imposed selection constraints. Our objective was to develop novel RNA enzymes by altering the selection constraints in a controlled manner. In this way we were able to expand the catalytic repertoire of RNA. Our long-range objective is to develop an RNA enzyme with RNA replicase activity. If such an enzyme had the ability to produce additional copies of itself, then RNA evolution would operate autonomously and the origin of life will have been realized in the laboratory.

Clay Caterpillars: A Tool for Ecology & Evolution Laboratories

ERIC Educational Resources Information Center

Barber, Nicholas A.

2012-01-01

I present a framework for ecology and evolution laboratory exercises using artificial caterpillars made from modeling clay. Students generate and test hypotheses about predation rates on caterpillars that differ in appearance or "behavior" to understand how natural selection by predators shapes distribution and physical characteristics of…

The Origin and Early Evolution of Membrane Proteins

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

2006-01-01

The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

Biophysical and structural considerations for protein sequence evolution

PubMed Central

2011-01-01

Background Protein sequence evolution is constrained by the biophysics of folding and function, causing interdependence between interacting sites in the sequence. However, current site-independent models of sequence evolutions do not take this into account. Recent attempts to integrate the influence of structure and biophysics into phylogenetic models via statistical/informational approaches have not resulted in expected improvements in model performance. This suggests that further innovations are needed for progress in this field. Results Here we develop a coarse-grained physics-based model of protein folding and binding function, and compare it to a popular informational model. We find that both models violate the assumption of the native sequence being close to a thermodynamic optimum, causing directional selection away from the native state. Sampling and simulation show that the physics-based model is more specific for fold-defining interactions that vary less among residue type. The informational model diffuses further in sequence space with fewer barriers and tends to provide less support for an invariant sites model, although amino acid substitutions are generally conservative. Both approaches produce sequences with natural features like dN/dS < 1 and gamma-distributed rates across sites. Conclusions Simple coarse-grained models of protein folding can describe some natural features of evolving proteins but are currently not accurate enough to use in evolutionary inference. This is partly due to improper packing of the hydrophobic core. We suggest possible improvements on the representation of structure, folding energy, and binding function, as regards both native and non-native conformations, and describe a large number of possible applications for such a model. PMID:22171550

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

PubMed Central

Biondi, Elisa; Lane, Joshua D.; Das, Debasis; Dasgupta, Saurja; Piccirilli, Joseph A.; Hoshika, Shuichi; Bradley, Kevin M.; Krantz, Bryan A.; Benner, Steven A.

2016-01-01

Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis. We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of ∼35 nM. These results illustrate the value of AEGIS-LIVE for those seeking to obtain receptors and ligands without the complexities of medicinal chemistry, and also challenge the biophysical community to develop new tools to analyze the spectroscopic signatures of new DNA folds that will emerge in synthetic genetic systems replacing standard DNA and RNA as platforms for LIVE. PMID:27701076

Directed molecular evolution to design advanced red fluorescent proteins.

PubMed

Subach, Fedor V; Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-11-29

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

Laboratory specimens and genetic privacy: evolution of legal theory.

PubMed

Lewis, Michelle Huckaby

2013-03-01

Although laboratory specimens are an important resource for biomedical research, controversy has arisen when research has been conducted without the knowledge or consent of the individuals who were the source of the specimens. This paper summarizes the most important litigation regarding the research use of laboratory specimens and traces the evolution of legal theory from property claims to claims related to genetic privacy interests. © 2013 American Society of Law, Medicine & Ethics, Inc.

Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis.

PubMed

Yang, Jin Kuk; Park, Min S; Waldo, Geoffrey S; Suh, Se Won

2003-01-21

One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140Tyr-153Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.

The protein-protein interface evolution acts in a similar way to antibody affinity maturation.

PubMed

Li, Bohua; Zhao, Lei; Wang, Chong; Guo, Huaizu; Wu, Lan; Zhang, Xunming; Qian, Weizhu; Wang, Hao; Guo, Yajun

2010-02-05

Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.

Simulating evolution of protein complexes through gene duplication and co-option.

PubMed

Haarsma, Loren; Nelesen, Serita; VanAndel, Ethan; Lamine, James; VandeHaar, Peter

2016-06-21

We present a model of the evolution of protein complexes with novel functions through gene duplication, mutation, and co-option. Under a wide variety of input parameters, digital organisms evolve complexes of 2-5 bound proteins which have novel functions but whose component proteins are not independently functional. Evolution of complexes with novel functions happens more quickly as gene duplication rates increase, point mutation rates increase, protein complex functional probability increases, protein complex functional strength increases, and protein family size decreases. Evolution of complexity is inhibited when the metabolic costs of making proteins exceeds the fitness gain of having functional proteins, or when point mutation rates get so large the functional proteins undergo deleterious mutations faster than new functional complexes can evolve. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation.

PubMed

Geller, Ron; Pechmann, Sebastian; Acevedo, Ashley; Andino, Raul; Frydman, Judith

2018-05-03

Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

PubMed Central

Reetz, Manfred T.

2004-01-01

A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the in vitro evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a “Darwinistic” process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted. PMID:15079053

Evolution of a mass spectrometry-grade protease with PTM-directed specificity.

PubMed

Tran, Duc T; Cavett, Valerie J; Dang, Vuong Q; Torres, Héctor L; Paegel, Brian M

2016-12-20

Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (k cat /K M = 6.9 × 10 5 M -1 ⋅s -1 ). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y 1 ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.

Computational design of chimeric protein libraries for directed evolution.

PubMed

Silberg, Jonathan J; Nguyen, Peter Q; Stevenson, Taylor

2010-01-01

The best approach for creating libraries of functional proteins with large numbers of nondisruptive amino acid substitutions is protein recombination, in which structurally related polypeptides are swapped among homologous proteins. Unfortunately, as more distantly related proteins are recombined, the fraction of variants having a disrupted structure increases. One way to enrich the fraction of folded and potentially interesting chimeras in these libraries is to use computational algorithms to anticipate which structural elements can be swapped without disturbing the integrity of a protein's structure. Herein, we describe how the algorithm Schema uses the sequences and structures of the parent proteins recombined to predict the structural disruption of chimeras, and we outline how dynamic programming can be used to find libraries with a range of amino acid substitution levels that are enriched in variants with low Schema disruption.

Accelerated rates of protein evolution in barley grain and pistil biased genes might be legacy of domestication.

PubMed

Shi, Tao; Dimitrov, Ivan; Zhang, Yinling; Tax, Frans E; Yi, Jing; Gou, Xiaoping; Li, Jia

2015-10-01

Traits related to grain and reproductive organs in grass crops have been under continuous directional selection during domestication. Barley is one of the oldest domesticated crops in human history. Thus genes associated with the grain and reproductive organs in barley may show evidence of dramatic evolutionary change. To understand how artificial selection contributes to protein evolution of biased genes in different barley organs, we used Digital Gene Expression analysis of six barley organs (grain, pistil, anther, leaf, stem and root) to identify genes with biased expression in specific organs. Pairwise comparisons of orthologs between barley and Brachypodium distachyon, as well as between highland and lowland barley cultivars mutually indicated that grain and pistil biased genes show relatively higher protein evolutionary rates compared with the median of all orthologs and other organ biased genes. Lineage-specific protein evolutionary rates estimation showed similar patterns with elevated protein evolution in barley grain and pistil biased genes, yet protein sequences generally evolve much faster in the lowland barley cultivar. Further functional annotations revealed that some of these grain and pistil biased genes with rapid protein evolution are related to nutrient biosynthesis and cell cycle/division. Our analyses provide insights into how domestication differentially shaped the evolution of genes specific to different organs of a crop species, and implications for future functional studies of domestication genes.

Amino acid Alphabet Size in Protein Evolution Experiments: Better to Search a Small library Thoroughly or a Large Library Sparsely?

PubMed Central

Muñoz, Enrique

2015-01-01

We compare the results obtained from searching a smaller library thoroughly versus searching a more diverse, larger library sparsely. We study protein evolution with reduced amino acid alphabets, by simulating directed evolution experiments at three different alphabet sizes: 20, 5 and 2. We employ a physical model for evolution, the generalized NK model, that has proved successful in modeling protein evolution, antibody evolution, and T cell selection. We find that antibodies with higher affinity are found by searching a library with a larger alphabet sparsely than by searching a smaller library thoroughly, even with well-designed reduced libraries. We find ranked amino acid usage frequencies in agreement with observations of the CDR-H3 variable region of human antibodies. PMID:18375453

Evolution of synthetic signaling scaffolds by recombination of modular protein domains.

PubMed

Lai, Andicus; Sato, Paloma M; Peisajovich, Sergio G

2015-06-19

Signaling scaffolds are proteins that interact via modular domains with multiple partners, regulating signaling networks in space and time and providing an ideal platform from which to alter signaling functions. However, to better exploit scaffolds for signaling engineering, it is necessary to understand the full extent of their modularity. We used a directed evolution approach to identify, from a large library of randomly shuffled protein interaction domains, variants capable of rescuing the signaling defect of a yeast strain in which Ste5, the scaffold in the mating pathway, had been deleted. After a single round of selection, we identified multiple synthetic scaffold variants with diverse domain architectures, able to mediate mating pathway activation in a pheromone-dependent manner. The facility with which this signaling network accommodates changes in scaffold architecture suggests that the mating signaling complex does not possess a single, precisely defined geometry into which the scaffold has to fit. These relaxed geometric constraints may facilitate the evolution of signaling networks, as well as their engineering for applications in synthetic biology.

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

PubMed Central

Gielen, Fabrice; Hours, Raphaelle; Emond, Stephane; Fischlechner, Martin; Schell, Ursula

2016-01-01

Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows. PMID:27821774

Evolution of ribosomal proteins in Enterobacteriaceae.

PubMed Central

Hori, H; Osawa, S

1978-01-01

The evolution of ribosomal proteins of about 70 bacterial strains belonging to the family Enterobacteriaceae has been studied by use of previously reported data (S. Osawa, T. Itoh, and E. Otaka, J. Bacteriol. 107:168-178, 1971) and those obtained in this paper. The proximity of the bacteria was quantified by co-chromatographing the differentially labeled ribosomal proteins from two strains on a column of carboxymethyl cellulose in various combinations. The were then classified into 12 groups (=species?) according to their ribosomal protein compositions and were placed in a phylogenic tree. PMID:346556

Witnessing Evolution First Hand: A K-12 Laboratory Exercise in Genetics & Evolution Using "Drosophila"

ERIC Educational Resources Information Center

Heil, Caiti S. S.; Manzano-Winkler, Brenda; Hunter, Mika J.; Noor, Juliet K. F.; Noor, Mohamed A. F.

2013-01-01

We present a laboratory exercise that leverages student interest in genetics to observe and understand evolution by natural selection. Students begin with white-eyed fruit fly populations, to which they introduce a single advantageous variant (one male with red eyes). The superior health and vision associated with having the red-eye-color allele…

Automating Microbial Directed Evolution For Bioengineering Applications

NASA Astrophysics Data System (ADS)

Lee, A.; Demachkie, I. S.; Sardesh, N.; Arismendi, D.; Ouandji, C.; Wang, J.; Blaich, J.; Gentry, D.

2016-12-01

From a micro-biology perspective, directed evolution is a technique that uses controlled environmental pressures to select for a desired phenotype. Directed evolution has the distinct advantage over rational design of not needing extensive knowledge of the genome or pathways associated with a microorganism to induce phenotypes. However, there are currently limitations to the applicability of this technique including being time-consuming, error-prone, and dependent on existing assays that may lack selectivity for the given phenotype. The AADEC (Autonomous Adaptive Directed Evolution Chamber) system is a proof-of-concept instrument to automate and improve the technique such that directed evolution can be used more effectively as a general bioengineering tool. A series of tests using the automated system and comparable by-hand survival assay measurements have been carried out using UV-C radiation and Escherichia coli cultures in order to demonstrate the advantages of the AADEC versus traditional implementations of directed evolution such as random mutagenesis. AADEC uses UV-C exposure as both a source of environmental stress and mutagenesis, so in order to evaluate the UV-C tolerance obtained from the cultures, a manual UV-C exposure survival assay was developed alongside the device to compare the survival fractions at a fixed dosage. This survival assay involves exposing E.coli to UV-C radiation using a custom-designed exposure hood to control the flux and dose. Surviving cells are counted then transferred to the next iteration and so on for several iterations to calculate the survival fractions for each exposure iteration. This survival assay primarily serves as a baseline for the AADEC device, allowing quantification of the differences between the AADEC system over the manual approach. The primary data of comparison is survival fractions; this is obtained by optical density and plate counts in the manual assay and by optical density growth curve fits pre- and post

Position Matters: Network Centrality Considerably Impacts Rates of Protein Evolution in the Human Protein–Protein Interaction Network

PubMed Central

Feyertag, Felix; Chakraborty, Sandip

2017-01-01

Abstract The proteins of any organism evolve at disparate rates. A long list of factors affecting rates of protein evolution have been identified. However, the relative importance of each factor in determining rates of protein evolution remains unresolved. The prevailing view is that evolutionary rates are dominantly determined by gene expression, and that other factors such as network centrality have only a marginal effect, if any. However, this view is largely based on analyses in yeasts, and accurately measuring the importance of the determinants of rates of protein evolution is complicated by the fact that the different factors are often correlated with each other, and by the relatively poor quality of available functional genomics data sets. Here, we use correlation, partial correlation and principal component regression analyses to measure the contributions of several factors to the variability of the rates of evolution of human proteins. For this purpose, we analyzed the entire human protein–protein interaction data set and the human signal transduction network—a network data set of exceptionally high quality, obtained by manual curation, which is expected to be virtually free from false positives. In contrast with the prevailing view, we observe that network centrality (measured as the number of physical and nonphysical interactions, betweenness, and closeness) has a considerable impact on rates of protein evolution. Surprisingly, the impact of centrality on rates of protein evolution seems to be comparable, or even superior according to some analyses, to that of gene expression. Our observations seem to be independent of potentially confounding factors and from the limitations (biases and errors) of interactomic data sets. PMID:28854629

[Structure and evolution of the eukaryotic FANCJ-like proteins].

PubMed

Wuhe, Jike; Zefeng, Wu; Sanhong, Fan; Xuguang, Xi

2015-02-01

The FANCJ-like protein family is a class of ATP-dependent helicases that can catalytically unwind duplex DNA along the 5'-3' direction. It is involved in the processes of DNA damage repair, homologous recombination and G-quadruplex DNA unwinding, and plays a critical role in maintaining genome integrity. In this study, we systemically analyzed FNACJ-like proteins from 47 eukaryotic species and discussed their sequences diversity, origin and evolution, motif organization patterns and spatial structure differences. Four members of FNACJ-like proteins, including XPD, CHL1, RTEL1 and FANCJ, were found in eukaryotes, but some of them were seriously deficient in most fungi and some insects. For example, the Zygomycota fungi lost RTEL1, Basidiomycota and Ascomycota fungi lost RTEL1 and FANCJ, and Diptera insect lost FANCJ. FANCJ-like proteins contain canonical motor domains HD1 and HD2, and the HD1 domain further integrates with three unique domains Fe-S, Arch and Extra-D. Fe-S and Arch domains are relatively conservative in all members of the family, but the Extra-D domain is lost in XPD and differs from one another in rest members. There are 7, 10 and 2 specific motifs found from the three unique domains respectively, while 5 and 12 specific motifs are found from HD1 and HD2 domains except the conserved motifs reported previously. By analyzing the arrangement pattern of these specific motifs, we found that RTEL1 and FANCJ are more closer and share two specific motifs Vb2 and Vc in HD2 domain, which are likely related with their G-quadruplex DNA unwinding activity. The evidence of evolution showed that FACNJ-like proteins were originated from a helicase, which has a HD1 domain inserted by extra Fe-S domain and Arch domain. By three continuous gene duplication events and followed specialization, eukaryotes finally possessed the current four members of FANCJ-like proteins.

Rational evolutionary design: the theory of in vitro protein evolution.

PubMed

Voigt, C A; Kauffman, S; Wang, Z G

2000-01-01

Directed evolution uses a combination of powerful search techniques to generate proteins with improved properties. Part of the success is due to the stochastic element of random mutagenesis; improvements can be made without a detailed description of the complex interactions that constitute function or stability. However, optimization is not a conglomeration of random processes. Rather, it requires both knowledge of the system that is being optimized and a logical series of techniques that best explores the pathways of evolution (Eigen et al., 1988). The weighing of parameters associated with mutation, recombination, and screening to achieve the maximum fitness improvement is the beginning of rational evolutionary design. The optimal mutation rate is strongly influenced by the finite number of mutants that can be screened. A smooth fitness landscape implies that many mutations can be accumulated without disrupting the fitness. This has the effect of lowering the required library size to sample a higher mutation rate. As the sequence ascends the fitness landscape, the optimal mutation rate decreases as the probability of discovering improved mutations also decreases. Highly coupled regions require that many mutations be simultaneously made to generate a positive mutant. Therefore, positive mutations are discovered at uncoupled positions as the fitness of the parent increases. The benefit of recombination is twofold: it combines good mutations and searches more sequence space in a meaningful way. Recombination is most beneficial when the number of mutants that can be screened is limited and the landscape is of an intermediate ruggedness. The structure of schema in proteins leads to the conclusion that many cut points are required. The number of parents and their sequence identity are determined by the balance between exploration and exploitation. Many disparate parents can explore more space, but at the risk of losing information. The required screening effort is

Evolution of genetic architecture under directional selection.

PubMed

Hansen, Thomas F; Alvarez-Castro, José M; Carter, Ashley J R; Hermisson, Joachim; Wagner, Günter P

2006-08-01

We investigate the multilinear epistatic model under mutation-limited directional selection. We confirm previous results that only directional epistasis, in which genes on average reinforce or diminish each other's effects, contribute to the initial evolution of mutational effects. Thus, either canalization or decanalization can occur under directional selection, depending on whether positive or negative epistasis is prevalent. We then focus on the evolution of the epistatic coefficients themselves. In the absence of higher-order epistasis, positive pairwise epistasis will tend to weaken relative to additive effects, while negative pairwise epistasis will tend to become strengthened. Positive third-order epistasis will counteract these effects, while negative third-order epistasis will reinforce them. More generally, gene interactions of all orders have an inherent tendency for negative changes under directional selection, which can only be modified by higher-order directional epistasis. We identify three types of nonadditive quasi-equilibrium architectures that, although not strictly stable, can be maintained for an extended time: (1) nondirectional epistatic architectures; (2) canalized architectures with strong epistasis; and (3) near-additive architectures in which additive effects keep increasing relative to epistasis.

Mean protein evolutionary distance: a method for comparative protein evolution and its application.

PubMed

Wise, Michael J

2013-01-01

Proteins are under tight evolutionary constraints, so if a protein changes it can only do so in ways that do not compromise its function. In addition, the proteins in an organism evolve at different rates. Leveraging the history of patristic distance methods, a new method for analysing comparative protein evolution, called Mean Protein Evolutionary Distance (MeaPED), measures differential resistance to evolutionary pressure across viral proteomes and is thereby able to point to the proteins' roles. Different species' proteomes can also be compared because the results, consistent across virus subtypes, concisely reflect the very different lifestyles of the viruses. The MeaPED method is here applied to influenza A virus, hepatitis C virus, human immunodeficiency virus (HIV), dengue virus, rotavirus A, polyomavirus BK and measles, which span the positive and negative single-stranded, doubled-stranded and reverse transcribing RNA viruses, and double-stranded DNA viruses. From this analysis, host interaction proteins including hemagglutinin (influenza), and viroporins agnoprotein (polyomavirus), p7 (hepatitis C) and VPU (HIV) emerge as evolutionary hot-spots. By contrast, RNA-directed RNA polymerase proteins including L (measles), PB1/PB2 (influenza) and VP1 (rotavirus), and internal serine proteases such as NS3 (dengue and hepatitis C virus) emerge as evolutionary cold-spots. The hot spot influenza hemagglutinin protein is contrasted with the related cold spot H protein from measles. It is proposed that evolutionary cold-spot proteins can become significant targets for second-line anti-viral therapeutics, in cases where front-line vaccines are not available or have become ineffective due to mutations in the hot-spot, generally more antigenically exposed proteins. The MeaPED package is available from www.pam1.bcs.uwa.edu.au/~michaelw/ftp/src/meaped.tar.gz.

Laboratory Directed Research and Development FY-15 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pillai, Rekha Sukamar

The Laboratory Directed Research and Development (LDRD) Program at Idaho National Laboratory (INL) reports its status to the U.S. Department of Energy (DOE) by March of each year. The program operates under the authority of DOE Order 413.2B, “Laboratory Directed Research and Development” (April 19, 2006), which establishes DOE’s requirements for the program while providing the laboratory director broad flexibility for program implementation. LDRD funds are obtained through a charge to all INL programs. This report includes summaries of all INL LDRD research activities supported during Fiscal Year (FY) 2015.

New Techniques for Ancient Proteins: Direct Coupling Analysis Applied on Proteins Involved in Iron Sulfur Cluster Biogenesis

PubMed Central

Fantini, Marco; Malinverni, Duccio; De Los Rios, Paolo; Pastore, Annalisa

2017-01-01

Direct coupling analysis (DCA) is a powerful statistical inference tool used to study protein evolution. It was introduced to predict protein folds and protein-protein interactions, and has also been applied to the prediction of entire interactomes. Here, we have used it to analyze three proteins of the iron-sulfur biogenesis machine, an essential metabolic pathway conserved in all organisms. We show that DCA can correctly reproduce structural features of the CyaY/frataxin family (a protein involved in the human disease Friedreich's ataxia) despite being based on the relatively small number of sequences allowed by its genomic distribution. This result gives us confidence in the method. Its application to the iron-sulfur cluster scaffold protein IscU, which has been suggested to function both as an ordered and a disordered form, allows us to distinguish evolutionary traces of the structured species, suggesting that, if present in the cell, the disordered form has not left evolutionary imprinting. We observe instead, for the first time, direct indications of how the protein can dimerize head-to-head and bind 4Fe4S clusters. Analysis of the alternative scaffold protein IscA provides strong support to a coordination of the cluster by a dimeric form rather than a tetramer, as previously suggested. Our analysis also suggests the presence in solution of a mixture of monomeric and dimeric species, and guides us to the prevalent one. Finally, we used DCA to analyze interactions between some of these proteins, and discuss the potentials and limitations of the method. PMID:28664160

Evolution of the SOUL Heme-Binding Protein Superfamily Across Eukarya.

PubMed

Fortunato, Antonio Emidio; Sordino, Paolo; Andreakis, Nikos

2016-06-01

SOUL homologs constitute a heme-binding protein superfamily putatively involved in heme and tetrapyrrole metabolisms associated with a number of physiological processes. Despite their omnipresence across the tree of life and the biochemical characterization of many SOUL members, their functional role and the evolutionary events leading to such remarkable protein repertoire still remain cryptic. To explore SOUL evolution, we apply a computational phylogenetic approach, including a relevant number of SOUL homologs, to identify paralog forms and reconstruct their genealogy across the tree of life and within species. In animal lineages, multiple gene duplication or loss events and paralog functional specializations underlie SOUL evolution from the dawn of ancestral echinoderm and mollusc SOUL forms. In photosynthetic organisms, SOUL evolution is linked to the endosymbiosis events leading to plastid acquisition in eukaryotes. Derivative features, such as the F2L peptide and BH3 domain, evolved in vertebrates and provided innovative functionality to support immune response and apoptosis. The evolution of elements such as the N-terminal protein domain DUF2358, the His42 residue, or the tetrapyrrole heme-binding site is modern, and their functional implications still unresolved. This study represents the first in-depth analysis of SOUL protein evolution and provides novel insights in the understanding of their obscure physiological role.

Mean Protein Evolutionary Distance: A Method for Comparative Protein Evolution and Its Application

PubMed Central

Wise, Michael J.

2013-01-01

Proteins are under tight evolutionary constraints, so if a protein changes it can only do so in ways that do not compromise its function. In addition, the proteins in an organism evolve at different rates. Leveraging the history of patristic distance methods, a new method for analysing comparative protein evolution, called Mean Protein Evolutionary Distance (MeaPED), measures differential resistance to evolutionary pressure across viral proteomes and is thereby able to point to the proteins’ roles. Different species’ proteomes can also be compared because the results, consistent across virus subtypes, concisely reflect the very different lifestyles of the viruses. The MeaPED method is here applied to influenza A virus, hepatitis C virus, human immunodeficiency virus (HIV), dengue virus, rotavirus A, polyomavirus BK and measles, which span the positive and negative single-stranded, doubled-stranded and reverse transcribing RNA viruses, and double-stranded DNA viruses. From this analysis, host interaction proteins including hemagglutinin (influenza), and viroporins agnoprotein (polyomavirus), p7 (hepatitis C) and VPU (HIV) emerge as evolutionary hot-spots. By contrast, RNA-directed RNA polymerase proteins including L (measles), PB1/PB2 (influenza) and VP1 (rotavirus), and internal serine proteases such as NS3 (dengue and hepatitis C virus) emerge as evolutionary cold-spots. The hot spot influenza hemagglutinin protein is contrasted with the related cold spot H protein from measles. It is proposed that evolutionary cold-spot proteins can become significant targets for second-line anti-viral therapeutics, in cases where front-line vaccines are not available or have become ineffective due to mutations in the hot-spot, generally more antigenically exposed proteins. The MeaPED package is available from www.pam1.bcs.uwa.edu.au/~michaelw/ftp/src/meaped.tar.gz. PMID:23613826

Protein interactions in 3D: from interface evolution to drug discovery.

PubMed

Winter, Christof; Henschel, Andreas; Tuukkanen, Anne; Schroeder, Michael

2012-09-01

Over the past 10years, much research has been dedicated to the understanding of protein interactions. Large-scale experiments to elucidate the global structure of protein interaction networks have been complemented by detailed studies of protein interaction interfaces. Understanding the evolution of interfaces allows one to identify convergently evolved interfaces which are evolutionary unrelated but share a few key residues and hence have common binding partners. Understanding interaction interfaces and their evolution is an important basis for pharmaceutical applications in drug discovery. Here, we review the algorithms and databases on 3D protein interactions and discuss in detail applications in interface evolution, drug discovery, and interface prediction. Copyright © 2012 Elsevier Inc. All rights reserved.

Directed evolution and synthetic biology applications to microbial systems.

PubMed

Bassalo, Marcelo C; Liu, Rongming; Gill, Ryan T

2016-06-01

Biotechnology applications require engineering complex multi-genic traits. The lack of knowledge on the genetic basis of complex phenotypes restricts our ability to rationally engineer them. However, complex phenotypes can be engineered at the systems level, utilizing directed evolution strategies that drive whole biological systems toward desired phenotypes without requiring prior knowledge of the genetic basis of the targeted trait. Recent developments in the synthetic biology field accelerates the directed evolution cycle, facilitating engineering of increasingly complex traits in biological systems. In this review, we summarize some of the most recent advances in directed evolution and synthetic biology that allows engineering of complex traits in microbial systems. Then, we discuss applications that can be achieved through engineering at the systems level. Copyright © 2016 Elsevier Ltd. All rights reserved.

Laboratory Directed Research and Development Program FY98

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, T.; Chartock, M.

1999-02-05

The Ernest Orlando Lawrence Berkeley National Laboratory (LBNL or Berkeley Lab) Laboratory Directed Research and Development Program FY 1998 report is compiled from annual reports submitted by principal investigators following the close of the fiscal year. This report describes the supported projects and summarizes their accomplishments. It constitutes a part of the Laboratory Directed Research and Development (LDRD) program planning and documentation process that includes an annual planning cycle, projection selection, implementation, and review. The LBNL LDRD program is a critical tool for directing the Laboratory's forefront scientific research capabilities toward vital, excellent, and emerging scientific challenges. The program providesmore » the resources for LBNL scientists to make rapid and significant contributions to critical national science and technology problems. The LDRD program also advances LBNL's core competencies, foundations, and scientific capability, and permits exploration of exciting new opportunities. All projects are work in forefront areas of science and technology. Areas eligible for support include the following: Advanced study of hypotheses, concepts, or innovative approaches to scientific or technical problems; Experiments and analyses directed toward ''proof of principle'' or early determination of the utility of new scientific ideas, technical concepts, or devices; and Conception and preliminary technical analyses of experimental facilities or devices.« less

Molecular evolution of cyclin proteins in animals and fungi

PubMed Central

2011-01-01

Background The passage through the cell cycle is controlled by complexes of cyclins, the regulatory units, with cyclin-dependent kinases, the catalytic units. It is also known that cyclins form several families, which differ considerably in primary structure from one eukaryotic organism to another. Despite these lines of evidence, the relationship between the evolution of cyclins and their function is an open issue. Here we present the results of our study on the molecular evolution of A-, B-, D-, E-type cyclin proteins in animals and fungi. Results We constructed phylogenetic trees for these proteins, their ancestral sequences and analyzed patterns of amino acid replacements. The analysis of infrequently fixed atypical amino acid replacements in cyclins evidenced that accelerated evolution proceeded predominantly during paralog duplication or after it in animals and fungi and that it was related to aromorphic changes in animals. It was shown also that evolutionary flexibility of cyclin function may be provided by consequential reorganization of regions on protein surface remote from CDK binding sites in animal and fungal cyclins and by functional differentiation of paralogous cyclins formed in animal evolution. Conclusions The results suggested that changes in the number and/or nature of cyclin-binding proteins may underlie the evolutionary role of the alterations in the molecular structure of cyclins and their involvement in diverse molecular-genetic events. PMID:21798004

The evolution of trade-offs under directional and correlational selection.

PubMed

Roff, Derek A; Fairbairn, Daphne J

2012-08-01

Using quantitative genetic theory, we develop predictions for the evolution of trade-offs in response to directional and correlational selection. We predict that directional selection favoring an increase in one trait in a trade-off will result in change in the intercept but not the slope of the trade-off function, with the mean value of the selected trait increasing and that of the correlated trait decreasing. Natural selection will generally favor an increase in some combination of trait values, which can be represented as directional selection on an index value. Such selection induces both directional and correlational selection on the component traits. Theory predicts that selection on an index value will also change the intercept but not the slope of the trade-off function but because of correlational selection, the direction of change in component traits may be in the same or opposite directions. We test these predictions using artificial selection on the well-established trade-off between fecundity and flight capability in the cricket, Gryllus firmus and compare the empirical results with a priori predictions made using genetic parameters from a separate half-sibling experiment. Our results support the predictions and illustrate the complexity of trade-off evolution when component traits are subject to both directional and correlational selection. © 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.

Practical aspects of protein co-evolution.

PubMed

Ochoa, David; Pazos, Florencio

2014-01-01

Co-evolution is a fundamental aspect of Evolutionary Theory. At the molecular level, co-evolutionary linkages between protein families have been used as indicators of protein interactions and functional relationships from long ago. Due to the complexity of the problem and the amount of genomic data required for these approaches to achieve good performances, it took a relatively long time from the appearance of the first ideas and concepts to the quotidian application of these approaches and their incorporation to the standard toolboxes of bioinformaticians and molecular biologists. Today, these methodologies are mature (both in terms of performance and usability/implementation), and the genomic information that feeds them large enough to allow their general application. This review tries to summarize the current landscape of co-evolution-based methodologies, with a strong emphasis on describing interesting cases where their application to important biological systems, alone or in combination with other computational and experimental approaches, allowed getting new insight into these.

Practical aspects of protein co-evolution

PubMed Central

Ochoa, David; Pazos, Florencio

2014-01-01

Co-evolution is a fundamental aspect of Evolutionary Theory. At the molecular level, co-evolutionary linkages between protein families have been used as indicators of protein interactions and functional relationships from long ago. Due to the complexity of the problem and the amount of genomic data required for these approaches to achieve good performances, it took a relatively long time from the appearance of the first ideas and concepts to the quotidian application of these approaches and their incorporation to the standard toolboxes of bioinformaticians and molecular biologists. Today, these methodologies are mature (both in terms of performance and usability/implementation), and the genomic information that feeds them large enough to allow their general application. This review tries to summarize the current landscape of co-evolution-based methodologies, with a strong emphasis on describing interesting cases where their application to important biological systems, alone or in combination with other computational and experimental approaches, allowed getting new insight into these. PMID:25364721

A single determinant dominates the rate of yeast protein evolution.

PubMed

Drummond, D Allan; Raval, Alpan; Wilke, Claus O

2006-02-01

A gene's rate of sequence evolution is among the most fundamental evolutionary quantities in common use, but what determines evolutionary rates has remained unclear. Here, we carry out the first combined analysis of seven predictors (gene expression level, dispensability, protein abundance, codon adaptation index, gene length, number of protein-protein interactions, and the gene's centrality in the interaction network) previously reported to have independent influences on protein evolutionary rates. Strikingly, our analysis reveals a single dominant variable linked to the number of translation events which explains 40-fold more variation in evolutionary rate than any other, suggesting that protein evolutionary rate has a single major determinant among the seven predictors. The dominant variable explains nearly half the variation in the rate of synonymous and protein evolution. We show that the two most commonly used methods to disentangle the determinants of evolutionary rate, partial correlation analysis and ordinary multivariate regression, produce misleading or spurious results when applied to noisy biological data. We overcome these difficulties by employing principal component regression, a multivariate regression of evolutionary rate against the principal components of the predictor variables. Our results support the hypothesis that translational selection governs the rate of synonymous and protein sequence evolution in yeast.

Conservation of hot regions in protein-protein interaction in evolution.

PubMed

Hu, Jing; Li, Jiarui; Chen, Nansheng; Zhang, Xiaolong

2016-11-01

The hot regions of protein-protein interactions refer to the active area which formed by those most important residues to protein combination process. With the research development on protein interactions, lots of predicted hot regions can be discovered efficiently by intelligent computing methods, while performing biology experiments to verify each every prediction is hardly to be done due to the time-cost and the complexity of the experiment. This study based on the research of hot spot residue conservations, the proposed method is used to verify authenticity of predicted hot regions that using machine learning algorithm combined with protein's biological features and sequence conservation, though multiple sequence alignment, module substitute matrix and sequence similarity to create conservation scoring algorithm, and then using threshold module to verify the conservation tendency of hot regions in evolution. This research work gives an effective method to verify predicted hot regions in protein-protein interactions, which also provides a useful way to deeply investigate the functional activities of protein hot regions. Copyright © 2016. Published by Elsevier Inc.

A Model for Designing Adaptive Laboratory Evolution Experiments.

PubMed

LaCroix, Ryan A; Palsson, Bernhard O; Feist, Adam M

2017-04-15

The occurrence of mutations is a cornerstone of the evolutionary theory of adaptation, capitalizing on the rare chance that a mutation confers a fitness benefit. Natural selection is increasingly being leveraged in laboratory settings for industrial and basic science applications. Despite increasing deployment, there are no standardized procedures available for designing and performing adaptive laboratory evolution (ALE) experiments. Thus, there is a need to optimize the experimental design, specifically for determining when to consider an experiment complete and for balancing outcomes with available resources (i.e., laboratory supplies, personnel, and time). To design and to better understand ALE experiments, a simulator, ALEsim, was developed, validated, and applied to the optimization of ALE experiments. The effects of various passage sizes were experimentally determined and subsequently evaluated with ALEsim, to explain differences in experimental outcomes. Furthermore, a beneficial mutation rate of 10 -6.9 to 10 -8.4 mutations per cell division was derived. A retrospective analysis of ALE experiments revealed that passage sizes typically employed in serial passage batch culture ALE experiments led to inefficient production and fixation of beneficial mutations. ALEsim and the results described here will aid in the design of ALE experiments to fit the exact needs of a project while taking into account the resources required and will lower the barriers to entry for this experimental technique. IMPORTANCE ALE is a widely used scientific technique to increase scientific understanding, as well as to create industrially relevant organisms. The manner in which ALE experiments are conducted is highly manual and uniform, with little optimization for efficiency. Such inefficiencies result in suboptimal experiments that can take multiple months to complete. With the availability of automation and computer simulations, we can now perform these experiments in an optimized

Seed storage proteins as a system for teaching protein identification by mass spectrometry in biochemistry laboratory.

PubMed

Wilson, Karl A; Tan-Wilson, Anna

2013-01-01

Mass spectrometry (MS) has become an important tool in studying biological systems. One application is the identification of proteins and peptides by the matching of peptide and peptide fragment masses to the sequences of proteins in protein sequence databases. Often prior protein separation of complex protein mixtures by 2D-PAGE is needed, requiring more time and expertise than instructors of large laboratory classes can devote. We have developed an experimental module for our Biochemistry Laboratory course that engages students in MS-based protein identification following protein separation by one-dimensional SDS-PAGE, a technique that is usually taught in this type of course. The module is based on soybean seed storage proteins, a relatively simple mixture of proteins present in high levels in the seed, allowing the identification of the main protein bands by MS/MS and in some cases, even by peptide mass fingerprinting. Students can identify their protein bands using software available on the Internet, and are challenged to deduce post-translational modifications that have occurred upon germination. A collection of mass spectral data and tutorials that can be used as a stand-alone computer-based laboratory module were also assembled. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Tracing Primordial Protein Evolution through Structurally Guided Stepwise Segment Elongation*

PubMed Central

Watanabe, Hideki; Yamasaki, Kazuhiko; Honda, Shinya

2014-01-01

The understanding of how primordial proteins emerged has been a fundamental and longstanding issue in biology and biochemistry. For a better understanding of primordial protein evolution, we synthesized an artificial protein on the basis of an evolutionary hypothesis, segment-based elongation starting from an autonomously foldable short peptide. A 10-residue protein, chignolin, the smallest foldable polypeptide ever reported, was used as a structural support to facilitate higher structural organization and gain-of-function in the development of an artificial protein. Repetitive cycles of segment elongation and subsequent phage display selection successfully produced a 25-residue protein, termed AF.2A1, with nanomolar affinity against the Fc region of immunoglobulin G. AF.2A1 shows exquisite molecular recognition ability such that it can distinguish conformational differences of the same molecule. The structure determined by NMR measurements demonstrated that AF.2A1 forms a globular protein-like conformation with the chignolin-derived β-hairpin and a tryptophan-mediated hydrophobic core. Using sequence analysis and a mutation study, we discovered that the structural organization and gain-of-function emerged from the vicinity of the chignolin segment, revealing that the structural support served as the core in both structural and functional development. Here, we propose an evolutionary model for primordial proteins in which a foldable segment serves as the evolving core to facilitate structural and functional evolution. This study provides insights into primordial protein evolution and also presents a novel methodology for designing small sized proteins useful for industrial and pharmaceutical applications. PMID:24356963

Experimental evolution of protein–protein interaction networks

PubMed Central

Kaçar, Betül; Gaucher, Eric A.

2013-01-01

The modern synthesis of evolutionary theory and genetics has enabled us to discover underlying molecular mechanisms of organismal evolution. We know that in order to maximize an organism's fitness in a particular environment, individual interactions among components of protein and nucleic acid networks need to be optimized by natural selection, or sometimes through random processes, as the organism responds to changes and/or challenges in the environment. Despite the significant role of molecular networks in determining an organism's adaptation to its environment, we still do not know how such inter- and intra-molecular interactions within networks change over time and contribute to an organism's evolvability while maintaining overall network functions. One way to address this challenge is to identify connections between molecular networks and their host organisms, to manipulate these connections, and then attempt to understand how such perturbations influence molecular dynamics of the network and thus influence evolutionary paths and organismal fitness. In the present review, we discuss how integrating evolutionary history with experimental systems that combine tools drawn from molecular evolution, synthetic biology and biochemistry allow us to identify the underlying mechanisms of organismal evolution, particularly from the perspective of protein interaction networks. PMID:23849056

Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

PubMed Central

2012-01-01

Background Proteins convey the majority of biochemical and cellular activities in organisms. Over the course of evolution, proteins undergo normal sequence mutations as well as large scale mutations involving domain duplication and/or domain shuffling. These events result in the generation of new proteins and protein families. Processes that affect proteome evolution drive species diversity and adaptation. Herein, change over the course of metazoan evolution, as defined by birth/death and duplication/deletion events within protein families and domains, was examined using the proteomes of 9 metazoan and two outgroup species. Results In studying members of the three major metazoan groups, the vertebrates, arthropods, and nematodes, we found that the number of protein families increased at the majority of lineages over the course of metazoan evolution where the magnitude of these increases was greatest at the lineages leading to mammals. In contrast, the number of protein domains decreased at most lineages and at all terminal lineages. This resulted in a weak correlation between protein family birth and domain birth; however, the correlation between domain birth and domain member duplication was quite strong. These data suggest that domain birth and protein family birth occur via different mechanisms, and that domain shuffling plays a role in the formation of protein families. The ratio of protein family birth to protein domain birth (domain shuffling index) suggests that shuffling had a more demonstrable effect on protein families in nematodes and arthropods than in vertebrates. Through the contrast of high and low domain shuffling indices at the lineages of Trichinella spiralis and Gallus gallus, we propose a link between protein redundancy and evolutionary changes controlled by domain shuffling; however, the speed of adaptation among the different lineages was relatively invariant. Evaluating the functions of protein families that appeared or disappeared at the

Domain organizations of modular extracellular matrix proteins and their evolution.

PubMed

Engel, J

1996-11-01

Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

Molecular Evolution of Aminoacyl tRNA Synthetase Proteins in the Early History of Life

NASA Astrophysics Data System (ADS)

Fournier, Gregory P.; Andam, Cheryl P.; Alm, Eric J.; Gogarten, J. Peter

2011-12-01

Aminoacyl-tRNA synthetases (aaRS) consist of several families of functionally conserved proteins essential for translation and protein synthesis. Like nearly all components of the translation machinery, most aaRS families are universally distributed across cellular life, being inherited from the time of the Last Universal Common Ancestor (LUCA). However, unlike the rest of the translation machinery, aaRS have undergone numerous ancient horizontal gene transfers, with several independent events detected between domains, and some possibly involving lineages diverging before the time of LUCA. These transfers reveal the complexity of molecular evolution at this early time, and the chimeric nature of genomes within cells that gave rise to the major domains. Additionally, given the role of these protein families in defining the amino acids used for protein synthesis, sequence reconstruction of their pre-LUCA ancestors can reveal the evolutionary processes at work in the origin of the genetic code. In particular, sequence reconstructions of the paralog ancestors of isoleucyl- and valyl- RS provide strong empirical evidence that at least for this divergence, the genetic code did not co-evolve with the aaRSs; rather, both amino acids were already part of the genetic code before their cognate aaRSs diverged from their common ancestor. The implications of this observation for the early evolution of RNA-directed protein biosynthesis are discussed.

Evolution of a protein folding nucleus.

PubMed

Xia, Xue; Longo, Liam M; Sutherland, Mason A; Blaber, Michael

2016-07-01

The folding nucleus (FN) is a cryptic element within protein primary structure that enables an efficient folding pathway and is the postulated heritable element in the evolution of protein architecture; however, almost nothing is known regarding how the FN structurally changes as complex protein architecture evolves from simpler peptide motifs. We report characterization of the FN of a designed purely symmetric β-trefoil protein by ϕ-value analysis. We compare the structure and folding properties of key foldable intermediates along the evolutionary trajectory of the β-trefoil. The results show structural acquisition of the FN during gene fusion events, incorporating novel turn structure created by gene fusion. Furthermore, the FN is adjusted by circular permutation in response to destabilizing functional mutation. FN plasticity by way of circular permutation is made possible by the intrinsic C3 cyclic symmetry of the β-trefoil architecture, identifying a possible selective advantage that helps explain the prevalence of cyclic structural symmetry in the proteome. © 2015 The Protein Society.

A Laboratory Demonstration Illustrating Bioseparations Using Colorful Proteins

ERIC Educational Resources Information Center

Cohen, Theodore M.; Rohs, Amanda E.; Lefebvre, Brian G.

2008-01-01

A simple in class laboratory illustrating the principles of ion exchange chromatography as a bioseparation technique is described. A protein's isoelectric point as a driving force for ion exchange chromatography is easily demonstrated by using combinations of proteins with natural color or fluorescence, such as DsRed2, enhanced green fluorescent…

The Origin and Early Evolution of Membrane Proteins

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Schweighofer, Karl; Wilson, Michael A.

2005-01-01

Membrane proteins mediate functions that are essential to all cells. These functions include transport of ions, nutrients and waste products across cell walls, capture of energy and its transduction into the form usable in chemical reactions, transmission of environmental signals to the interior of the cell, cellular growth and cell volume regulation. In the absence of membrane proteins, ancestors of cell (protocells), would have had only very limited capabilities to communicate with their environment. Thus, it is not surprising that membrane proteins are quite common even in simplest prokaryotic cells. Considering that contemporary membrane channels are large and complex, both structurally and functionally, a question arises how their presumably much simpler ancestors could have emerged, perform functions and diversify in early protobiological evolution. Remarkably, despite their overall complexity, structural motifs in membrane proteins are quite simple, with a-helices being most common. This suggests that these proteins might have evolved from simple building blocks. To explain how these blocks could have organized into functional structures, we performed large-scale, accurate computer simulations of folding peptides at a water-membrane interface, their insertion into the membrane, self-assembly into higher-order structures and function. The results of these simulations, combined with analysis of structural and functional experimental data led to the first integrated view of the origin and early evolution of membrane proteins.

A Novel Laboratory Activity for Teaching about the Evolution of Multicellularity

ERIC Educational Resources Information Center

Ratcliff, William C.; Raney, Allison; Westreich, Sam; Cotner, Sehoya

2014-01-01

The evolution of complexity remains one of the most challenging topics in biology to teach effectively. We present a novel laboratory activity, modeled on a recent experimental breakthrough, in which students experimentally evolve simple multicellularity using single-celled yeast ("Saccharomyces cerevisiae"). By simply selecting for…

Cell-selfish modes of evolution and mutations directed after transcriptional bypass.

PubMed

Holmquist, Gerald P

2002-12-29

During transcription, prokaryotic and eukaryotic RNA polymerases bypass and misread (transcriptional mutagenesis) several classes of DNA lesions. For example, misreading of 8-OH-dG generates mRNAs containing G to T transversions. After translation, if the mutant protein briefly allowed the cell a growth-DNA replication advantage, then precocious DNA replication would bypass that unrepaired 8-OH-dG and misinsert dA opposite the directing DNA lesion with a higher probability than would be experienced for 8-OH-G lesions at other positions in otherwise identical neighboring cells. Such retromutations would have been tested for their imparted growth advantage as mRNA before they became heritable DNA mutations. The logical properties of a mode of evolution that utilizes directed-retromutagenesis were compared one by one with those of the standard neo-Darwinian mode. The retromutagenesis mode, while minimizing mutational load, is cell-selfish; fitness is for an immediate growth advantage rather than future reproductive potential. In prokaryotes, an evolutionary mode that involves standard Darwinian fitness testing of novel alleles in the genetic background of origin followed by clonal expansion also favors cell-selfish allele combinations when linkage disequilibrium is practiced. For metazoa and plants to have evolved organized tissues, cell-selfish modes of evolution represent systems-poisons that must be totally suppressed. The feedback loops that allow evolution to be cell-serving in prokaryotes are actively blocked in eukaryotes by traits that restrict fitness to future reproductive potential. These traits include (i) delay of fitness testing until after the mutation is made permanently heritable, (ii) diploidy to further delay fitness testing, (iii) segregation of somatic lines from germ lines, (iv) testing of novel alleles against randomized allele combinations constructed by obligate sex, and (v) obligate genetic death to insure that that the most basic systems

Laboratory Directed Research and Development Program FY 2006 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sjoreen, Terrence P

2007-04-01

The Oak Ridge National Laboratory (ORNL) Laboratory Directed Research and Development (LDRD) Program reports its status to the US Departmental of Energy (DOE) in March of each year. The program operates under the authority of DOE Order 413.2B, 'Laboratory Directed Research and Development' (April 19, 2006), which establishes DOE's requirements for the program while providing the Laboratory Director broad flexibility for program implementation. LDRD funds are obtained through a charge to all Laboratory programs. This report includes summaries all ORNL LDRD research activities supported during FY 2006. The associated FY 2006 ORNL LDRD Self-Assessment (ORNL/PPA-2007/2) provides financial data about themore » FY 2006 projects and an internal evaluation of the program's management process.« less

Laboratory Directed Research and Development FY 2000 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Ayat, R

This Annual Report provides an overview of the FY2000 Laboratory Directed Research and Development (LDRD) Program at Lawrence Livermore National Laboratory (LLNL) and presents a summary of the results achieved by each project during the year.

ScaffoldSeq: Software for characterization of directed evolution populations.

PubMed

Woldring, Daniel R; Holec, Patrick V; Hackel, Benjamin J

2016-07-01

ScaffoldSeq is software designed for the numerous applications-including directed evolution analysis-in which a user generates a population of DNA sequences encoding for partially diverse proteins with related functions and would like to characterize the single site and pairwise amino acid frequencies across the population. A common scenario for enzyme maturation, antibody screening, and alternative scaffold engineering involves naïve and evolved populations that contain diversified regions, varying in both sequence and length, within a conserved framework. Analyzing the diversified regions of such populations is facilitated by high-throughput sequencing platforms; however, length variability within these regions (e.g., antibody CDRs) encumbers the alignment process. To overcome this challenge, the ScaffoldSeq algorithm takes advantage of conserved framework sequences to quickly identify diverse regions. Beyond this, unintended biases in sequence frequency are generated throughout the experimental workflow required to evolve and isolate clones of interest prior to DNA sequencing. ScaffoldSeq software uniquely handles this issue by providing tools to quantify and remove background sequences, cluster similar protein families, and dampen the impact of dominant clones. The software produces graphical and tabular summaries for each region of interest, allowing users to evaluate diversity in a site-specific manner as well as identify epistatic pairwise interactions. The code and detailed information are freely available at http://research.cems.umn.edu/hackel. Proteins 2016; 84:869-874. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

1995 Laboratory-Directed Research and Development Annual report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cauffman, D.P.; Shoaf, D.L.; Hill, D.A.

1995-12-31

The Laboratory-Directed Research and Development Program (LDRD) is a key component of the discretionary research conducted by Lockheed Idaho Technologies Company (Lockheed Idaho) at the Idaho National Engineering Laboratory (INEL). The threefold purpose and goal of the LDRD program is to maintain the scientific and technical vitality of the INEL, respond to and support new technical opportunities, and enhance the agility and flexibility of the national laboratory and Lockheed Idaho to address the current and future missions of the Department of Energy.

A First-Principles Model of Early Evolution: Emergence of Gene Families, Species, and Preferred Protein Folds

PubMed Central

Zeldovich, Konstantin B; Chen, Peiqiu; Shakhnovich, Boris E; Shakhnovich, Eugene I

2007-01-01

In this work we develop a microscopic physical model of early evolution where phenotype—organism life expectancy—is directly related to genotype—the stability of its proteins in their native conformations—which can be determined exactly in the model. Simulating the model on a computer, we consistently observe the “Big Bang” scenario whereby exponential population growth ensues as soon as favorable sequence–structure combinations (precursors of stable proteins) are discovered. Upon that, random diversity of the structural space abruptly collapses into a small set of preferred proteins. We observe that protein folds remain stable and abundant in the population at timescales much greater than mutation or organism lifetime, and the distribution of the lifetimes of dominant folds in a population approximately follows a power law. The separation of evolutionary timescales between discovery of new folds and generation of new sequences gives rise to emergence of protein families and superfamilies whose sizes are power-law distributed, closely matching the same distributions for real proteins. On the population level we observe emergence of species—subpopulations that carry similar genomes. Further, we present a simple theory that relates stability of evolving proteins to the sizes of emerging genomes. Together, these results provide a microscopic first-principles picture of how first-gene families developed in the course of early evolution. PMID:17630830

A first-principles model of early evolution: emergence of gene families, species, and preferred protein folds.

PubMed

Zeldovich, Konstantin B; Chen, Peiqiu; Shakhnovich, Boris E; Shakhnovich, Eugene I

2007-07-01

In this work we develop a microscopic physical model of early evolution where phenotype--organism life expectancy--is directly related to genotype--the stability of its proteins in their native conformations-which can be determined exactly in the model. Simulating the model on a computer, we consistently observe the "Big Bang" scenario whereby exponential population growth ensues as soon as favorable sequence-structure combinations (precursors of stable proteins) are discovered. Upon that, random diversity of the structural space abruptly collapses into a small set of preferred proteins. We observe that protein folds remain stable and abundant in the population at timescales much greater than mutation or organism lifetime, and the distribution of the lifetimes of dominant folds in a population approximately follows a power law. The separation of evolutionary timescales between discovery of new folds and generation of new sequences gives rise to emergence of protein families and superfamilies whose sizes are power-law distributed, closely matching the same distributions for real proteins. On the population level we observe emergence of species--subpopulations that carry similar genomes. Further, we present a simple theory that relates stability of evolving proteins to the sizes of emerging genomes. Together, these results provide a microscopic first-principles picture of how first-gene families developed in the course of early evolution.

Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.

PubMed

Nyerges, Ákos; Csörgő, Bálint; Draskovits, Gábor; Kintses, Bálint; Szili, Petra; Ferenc, Györgyi; Révész, Tamás; Ari, Eszter; Nagy, István; Bálint, Balázs; Vásárhelyi, Bálint Márk; Bihari, Péter; Számel, Mónika; Balogh, Dávid; Papp, Henrietta; Kalapis, Dorottya; Papp, Balázs; Pál, Csaba

2018-06-19

Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology. Copyright © 2018 the Author(s). Published by PNAS.

A 13-Week Research-Based Biochemistry Laboratory Curriculum

ERIC Educational Resources Information Center

Lefurgy, Scott T.; Mundorff, Emily C.

2017-01-01

Here, we present a 13-week research-based biochemistry laboratory curriculum designed to provide the students with the experience of engaging in original research while introducing foundational biochemistry laboratory techniques. The laboratory experience has been developed around the directed evolution of an enzyme chosen by the instructor, with…

Playing evolution in the laboratory: From the first major evolutionary transition to global warming

NASA Astrophysics Data System (ADS)

Fragata, Inês; Simões, Pedro; Matos, Margarida; Szathmáry, Eörs; Santos, Mauro

2018-05-01

Experimental evolution allows testing hypotheses derived from theory or from observed patterns in nature. We have designed a droplet-based microfluidic “evolution machine” to test how transient compartmentalization (“trait-groups”) of independent molecular replicators (likely a critical step in the origin of life) could have prevented the spread of parasitic mutants; that is, inactive RNAs that have been reported to spoil a system of free replicators. In remarkable agreement with the theory, we show that this simple population structure was sufficient to prevent takeover by inactive RNAs. A more complex scenario arises when we use experimental evolution to test field-derived hypotheses; for instance, the idea that temperature is driving genetic spatiotemporal patterns of climate change. In the fly Drosophila subobscura, latitudinal clines in gene arrangement frequencies occur worldwide, and more equatorial gene arrangements are becoming more frequent at higher latitudes as a correlated response to climate change. However, the evolution at different constant temperatures in the laboratory was not consistent with patterns in nature, suggesting some limitations of experimental evolution. Finally, also in D. subobscura, we show that repeatability in experimental evolution is staggeringly consistent for life history traits, making evolution quite predictable and suggesting that laboratory selection can quickly erase differences between populations. Yet, the genetic paths used to attain the same adaptive phenotypes are complex and unpredictable. Contribution to the Focus Issue Evolutionary Modeling and Experimental Evolution edited by José Cuesta, Joachim Krug and Susanna Manrubia.

Directed evolution of polymerase function by compartmentalized self-replication.

PubMed

Ghadessy, F J; Ong, J L; Holliger, P

2001-04-10

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.

New Measurement for Correlation of Co-evolution Relationship of Subsequences in Protein.

PubMed

Gao, Hongyun; Yu, Xiaoqing; Dou, Yongchao; Wang, Jun

2015-12-01

Many computational tools have been developed to measure the protein residues co-evolution. Most of them only focus on co-evolution for pairwise residues in a protein sequence. However, number of residues participate in co-evolution might be multiple. And some co-evolved residues are clustered in several distinct regions in primary structure. Therefore, the co-evolution among the adjacent residues and the correlation between the distinct regions offer insights into function and evolution of the protein and residues. Subsequence is used to represent the adjacent multiple residues in one distinct region. In the paper, co-evolution relationship in each subsequence is represented by mutual information matrix (MIM). Then, Pearson's correlation coefficient: R value is developed to measure the similarity correlation of two MIMs. MSAs from Catalytic Data Base (Catalytic Site Atlas, CSA) are used for testing. R value characterizes a specific class of residues. In contrast to individual pairwise co-evolved residues, adjacent residues without high individual MI values are found since the co-evolved relationship among them is similar to that among another set of adjacent residues. These subsequences possess some flexibility in the composition of side chains, such as the catalyzed environment.

Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

USDA-ARS?s Scientific Manuscript database

Understanding proteome evolution is important for deciphering processes that drive species diversity and adaptation. Herein, the dynamics of change in protein families and protein domains over the course of metazoan evolution was explored. Change, as defined by birth/death and duplication/deletion ...

The Evolution of Human Cells in Terms of Protein Innovation

PubMed Central

Sardar, Adam J.; Oates, Matt E.; Fang, Hai; Forrest, Alistair R.R.; Kawaji, Hideya; Gough, Julian; Rackham, Owen J.L.

2014-01-01

Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type–specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type–specific domain architectures. PMID:24692656

Tailoring in vitro evolution for protein affinity or stability

PubMed Central

Jermutus, Lutz; Honegger, Annemarie; Schwesinger, Falk; Hanes, Jozef; Plückthun, Andreas

2001-01-01

We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (koff at 4°C of 10−4 s−1) was improved 30-fold by the use of off-rate selections over a period of several days. As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs. We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol. Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure. Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible. PMID:11134506

Polishing the craft of genetic diversity creation in directed evolution.

PubMed

Tee, Kang Lan; Wong, Tuck Seng

2013-12-01

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating "smart" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field. © 2013. Published by Elsevier Inc. All rights reserved.

The evolution of resistance genes in multi-protein plant resistance systems.

PubMed

Friedman, Aaron R; Baker, Barbara J

2007-12-01

The genomic perspective aids in integrating the analysis of single resistance (R-) genes into a higher order model of complex plant resistance systems. The majority of R-genes encode a class of proteins with nucleotide binding (NB) and leucine-rich repeat (LRR) domains. Several R-proteins act in multi-protein R-complexes that mediate interaction with pathogen effectors to induce resistance signaling. The complexity of these systems seems to have resulted from multiple rounds of plant-pathogen co-evolution. R-gene evolution is thought to be facilitated by the formation of R-gene clusters, which permit sequence exchanges via recombinatorial mispairing and generate high haplotypic diversity. This pattern of evolution may also generate diversity at other loci that contribute to the R-complex. The rate of recombination at R-clusters is not necessarily homogeneous or consistent over evolutionary time: recent evidence suggests that recombination at R-clusters is increased following pathogen infection, suggesting a mechanism that induces temporary genome instability in response to extreme stress. DNA methylation and chromatin modifications may allow this instability to be conditionally regulated and targeted to specific genome regions. Knowledge of natural R-gene evolution may contribute to strategies for artificial evolution of novel resistance specificities.

A Mutation Directs the Structural Switch of DNA Binding Proteins under Starvation to a Ferritin-like Protein Cage.

PubMed

Williams, Sunanda Margrett; Chandran, Anu Vijayakumari; Prakash, Sunita; Vijayan, Mamannamana; Chatterji, Dipankar

2017-09-05

Proteins of the ferritin family are ubiquitous in living organisms. With their spherical cage-like structures they are the iron storehouses in cells. Subfamilies of ferritins include 24-meric ferritins and bacterioferritins (maxiferritins), and 12-meric Dps (miniferritins). Dps safeguards DNA by direct binding, affording physical protection and safeguards from free radical-mediated damage by sequestering iron in its core. The maxiferritins can oxidize and store iron but cannot bind DNA. Here we show that a mutation at a critical interface in Dps alters its assembly from the canonical 12-mer to a ferritin-like 24-mer under crystallization. This structural switch was attributed to the conformational alteration of a highly conserved helical loop and rearrangement of the C-terminus. Our results demonstrate a novel concept of mutational switch between related protein subfamilies and corroborate the popular model for evolution by which subtle substitutions in an amino acid sequence lead to diversification among proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Laboratory Directed Research and Development Program FY2016 Annual Summary of Completed Projects

DOE Office of Scientific and Technical Information (OSTI.GOV)

None, None

ORNL FY 2016 Annual Summary of Laboratory Directed Research and Development Program (LDRD) Completed Projects. The Laboratory Directed Research and Development (LDRD) program at ORNL operates under the authority of DOE Order 413.2C, “Laboratory Directed Research and Development” (October 22, 2015), which establishes DOE’s requirements for the program while providing the Laboratory Director broad flexibility for program implementation. The LDRD program funds are obtained through a charge to all Laboratory programs. ORNL reports its status to DOE in March of each year.

Trends in global warming and evolution of matrix protein 2 family from influenza A virus.

PubMed

Yan, Shao-Min; Wu, Guang

2009-12-01

The global warming is an important factor affecting the biological evolution, and the influenza is an important disease that threatens humans with possible epidemics or pandemics. In this study, we attempted to analyze the trends in global warming and evolution of matrix protein 2 family from influenza A virus, because this protein is a target of anti-flu drug, and its mutation would have significant effect on the resistance to anti-flu drugs. The evolution of matrix protein 2 of influenza A virus from 1959 to 2008 was defined using the unpredictable portion of amino-acid pair predictability. Then the trend in this evolution was compared with the trend in the global temperature, the temperature in north and south hemispheres, and the temperature in influenza A virus sampling site, and species carrying influenza A virus. The results showed the similar trends in global warming and in evolution of M2 proteins although we could not correlate them at this stage of study. The study suggested the potential impact of global warming on the evolution of proteins from influenza A virus.

Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al, Hui-wang; Henderson, J. Nathan; Remington, S. James

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identifiedmore » dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 {angstrom} crystal structure (1 {angstrom}=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.« less

Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity

PubMed Central

Mack, Korrie L.; Shorter, James

2016-01-01

Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702

Directed evolution of stereoselective enzymes based on genetic selection as opposed to screening systems.

PubMed

Acevedo-Rocha, Carlos G; Agudo, Ruben; Reetz, Manfred T

2014-12-10

Directed evolution of stereoselective enzymes provides a means to generate useful biocatalysts for asymmetric transformations in organic chemistry and biotechnology. Almost all of the numerous examples reported in the literature utilize high-throughput screening systems based on suitable analytical techniques. Since the screening step is the bottleneck of the overall procedure, researchers have considered the use of genetic selection systems as an alternative to screening. In principle, selection would be the most elegant and efficient approach because it is based on growth advantage of host cells harboring stereoselective mutants, but devising such selection systems is very challenging. They must be designed so that the host organism profits from the presence of an enantioselective variant. Progress in this intriguing research area is summarized in this review, which also includes some examples of display systems designed for enantioselectivity as assayed by fluorescence-activated cell sorting (FACS). Although the combination of display systems and FACS is a powerful approach, we also envision innovative ideas combining metabolic engineering and genetic selection systems with protein directed evolution for the development of highly selective and efficient biocatalysts. Copyright © 2014 Elsevier B.V. All rights reserved.

1999 LDRD Laboratory Directed Research and Development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rita Spencer; Kyle Wheeler

This is the FY 1999 Progress Report for the Laboratory Directed Research and Development (LDRD) Program at Los Alamos National Laboratory. It gives an overview of the LDRD Program, summarizes work done on individual research projects, relates the projects to major Laboratory program sponsors, and provides an index to the principal investigators. Project summaries are grouped by their LDRD component: Competency Development, Program Development, and Individual Projects. Within each component, they are further grouped into nine technical categories: (1) materials science, (2) chemistry, (3) mathematics and computational science, (4) atomic, molecular, optical, and plasma physics, fluids, and particle beams, (5)more » engineering science, (6) instrumentation and diagnostics, (7) geoscience, space science, and astrophysics, (8) nuclear and particle physics, and (9) bioscience.« less

Engineering Biosynthesis of Non-ribosomal Peptides and Polyketides by Directed Evolution.

PubMed

Rui, Zhe; Zhang, Wenjun

2016-01-01

Non-ribosomal peptides (NRPs) and polyketides (PKs) play key roles in pharmaceutical industry due to their promising biological activities. The structural complexity of NRPs and PKs, however, creates significant synthetic challenges for producing these natural products and their analogues by purely chemical means. Alternatively, difficult syntheses can be achieved by using biosynthetic enzymes with improved efficiency and altered selectivity that are acquired from directed evolution. Key to the successful directed evolution is the methodology of screening/selection. This review summarizes the screening/selection strategies that have been employed to improve or modify the functions of non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), in the hope of triggering the wide adoption of the directed evolution approaches in the engineered biosynthesis of NRPs and PKs for drug discovery.

Tracking the origin and divergence of cholinesterases and neuroligins: the evolution of synaptic proteins.

PubMed

Lenfant, Nicolas; Hotelier, Thierry; Bourne, Yves; Marchot, Pascale; Chatonnet, Arnaud

2014-07-01

A cholinesterase activity can be found in all kingdoms of living organism, yet cholinesterases involved in cholinergic transmission appeared only recently in the animal phylum. Among various proteins homologous to cholinesterases, one finds neuroligins. These proteins, with an altered catalytic triad and no known hydrolytic activity, display well-identified cell adhesion properties. The availability of complete genomes of a few metazoans provides opportunities to evaluate when these two protein families emerged during evolution. In bilaterian animals, acetylcholinesterase co-localizes with proteins of cholinergic synapses while neuroligins co-localize and may interact with proteins of excitatory glutamatergic or inhibitory GABAergic/glycinergic synapses. To compare evolution of the cholinesterases and neuroligins with other proteins involved in the architecture and functioning of synapses, we devised a method to search for orthologs of these partners in genomes of model organisms representing distinct stages of metazoan evolution. Our data point to a progressive recruitment of synaptic components during evolution. This finding may shed light on the common or divergent developmental regulation events involved into the setting and maintenance of the cholinergic versus glutamatergic and GABAergic/glycinergic synapses.

Characterizing Microbe-Environment Interactions Through Experimental Evolution: The Autonomous Adaptive Directed Evolution Chamber

NASA Astrophysics Data System (ADS)

Ibanez, C. R.; Blaich, J.; Owyang, S.; Storrs, A.; Moffet, A.; Wong, N.; Zhou, J.; Gentry, D.

2015-12-01

We are developing a laboratory system for studying micro- to meso-scale interactions between microorganisms and their physicochemical environments. The Autonomous Adaptive Directed Evolution Chamber (AADEC) cultures microorganisms in controlled,small-scale geochemical environments. It observes corresponding microbial interactions to these environments and has the ability to adjust thermal, chemical, and other parameters in real time in response to these interactions. In addition to the sensed data, the system allows the generation of time-resolved ecological, genomic, etc. samples on the order of microbial generations. The AADEC currently houses cultures in liquid media and controls UVC radiation, heat exposure, and nutrient supply. In a proof-of-concept experimental evolution application, it can increase UVC radiation resistance of Escherichia coli cultures by iteratively exposing them to UVC and allowing the surviving cells to regrow. A baseline characterization generated a million fold resistance increase. This demonstration uses a single-well growth chamber prototype, but it was limited by scalability. We have expanded upon this system by implementing a microwell plate compatible fluidics system and sensor housing. This microwell plate system increases the diversity of microbial interactions seen in response to the geochemical environments generated by the system, allowing greater control over individual cultures' environments and detection of rarer events. The custom microfluidic card matches the footprint of a standard microwell plate. This card enables controllable fluid flow between wells and introduces multiple separate exposure and sensor chambers, increasing the variety of sensors compatible with the system. This gives the device control over scale and the interconnectedness of environments within the system. The increased controllability of the multiwell system provides a platform for implementing machine learning algorithms that will autonomously

Selection platforms for directed evolution in synthetic biology

PubMed Central

Tizei, Pedro A.G.; Csibra, Eszter; Torres, Leticia; Pinheiro, Vitor B.

2016-01-01

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules–gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function–be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code. PMID:27528765

Evolution-Based Functional Decomposition of Proteins

PubMed Central

Rivoire, Olivier; Reynolds, Kimberly A.; Ranganathan, Rama

2016-01-01

The essential biological properties of proteins—folding, biochemical activities, and the capacity to adapt—arise from the global pattern of interactions between amino acid residues. The statistical coupling analysis (SCA) is an approach to defining this pattern that involves the study of amino acid coevolution in an ensemble of sequences comprising a protein family. This approach indicates a functional architecture within proteins in which the basic units are coupled networks of amino acids termed sectors. This evolution-based decomposition has potential for new understandings of the structural basis for protein function. To facilitate its usage, we present here the principles and practice of the SCA and introduce new methods for sector analysis in a python-based software package (pySCA). We show that the pattern of amino acid interactions within sectors is linked to the divergence of functional lineages in a multiple sequence alignment—a model for how sector properties might be differentially tuned in members of a protein family. This work provides new tools for studying proteins and for generally testing the concept of sectors as the principal units of function and adaptive variation. PMID:27254668

Directionality theory and the evolution of body size.

PubMed

Demetrius, L

2000-12-07

Directionality theory, a dynamic theory of evolution that integrates population genetics with demography, is based on the concept of evolutionary entropy, a measure of the variability in the age of reproducing individuals in a population. The main tenets of the theory are three principles relating the response to the ecological constraints a population experiences, with trends in entropy as the population evolves under mutation and natural selection. (i) Stationary size or fluctuations around a stationary size (bounded growth): a unidirectional increase in entropy; (ii) prolonged episodes of exponential growth (unbounded growth), large population size: a unidirectional decrease in entropy; and (iii) prolonged episodes of exponential growth (unbounded growth), small population size: random, non-directional change in entropy. We invoke these principles, together with an allometric relationship between entropy, and the morphometric variable body size, to provide evolutionary explanations of three empirical patterns pertaining to trends in body size, namely (i) Cope's rule, the tendency towards size increase within phyletic lineages; (ii) the island rule, which pertains to changes in body size that occur as species migrate from mainland populations to colonize island habitats; and (iii) Bergmann's rule, the tendency towards size increase with increasing latitude. The observation that these ecotypic patterns can be explained in terms of the directionality principles for entropy underscores the significance of evolutionary entropy as a unifying concept in forging a link between micro-evolution, the dynamics of gene frequency change, and macro-evolution, dynamic changes in morphometric variables.

Unintended Laboratory-Driven Evolution Reveals Genetic Requirements for Biofilm Formation by Desulfovibrio vulgaris Hildenborough

DOE PAGES

De León, Kara B.; Zane, Grant M.; Trotter, Valentine V.; ...

2017-10-17

Biofilms of sulfate-reducing bacteria (SRB) are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here in this paper, we show that two supposedly identical wild-type cultures of the SRBDesulfovibrio vulgarisHildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS), was sufficientmore » to eliminate biofilm formation inD. vulgarisHildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient) was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered. The growth of bacteria attached to a surface (i.e., biofilm), specifically biofilms of sulfate-reducing bacteria, has a profound impact on the economy of developed nations due to steel and concrete corrosion in industrial pipelines and processing facilities. Furthermore, the presence of sulfate-reducing bacteria in oil wells causes oil souring from sulfide production

Unintended Laboratory-Driven Evolution Reveals Genetic Requirements for Biofilm Formation by Desulfovibrio vulgaris Hildenborough

DOE Office of Scientific and Technical Information (OSTI.GOV)

De León, Kara B.; Zane, Grant M.; Trotter, Valentine V.

Biofilms of sulfate-reducing bacteria (SRB) are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here in this paper, we show that two supposedly identical wild-type cultures of the SRBDesulfovibrio vulgarisHildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS), was sufficientmore » to eliminate biofilm formation inD. vulgarisHildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient) was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered. The growth of bacteria attached to a surface (i.e., biofilm), specifically biofilms of sulfate-reducing bacteria, has a profound impact on the economy of developed nations due to steel and concrete corrosion in industrial pipelines and processing facilities. Furthermore, the presence of sulfate-reducing bacteria in oil wells causes oil souring from sulfide production

ORNLs Laboratory Directed Research and Development Program FY 2009 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

None, None

2010-03-01

The Laboratory Directed Research and Development (LDRD) program at Oak Ridge National Laboratory (ORNL) reports its status to the U.S. Department of Energy (DOE) in March of each year. The program operates under the authority of DOE Order 413.2B, “Laboratory Directed Research and Development” (April 19, 2006), which establishes DOE’s requirements for the program while providing the Laboratory Director broad flexibility for program implementation. LDRD funds are obtained through a charge to all Laboratory programs. This report includes summaries all ORNL LDRD research activities supported during FY 2009. The associated FY 2009 ORNL LDRD Self-Assessment (ORNL/PPA-2010/2) provides financial data andmore » an internal evaluation of the program’s management process.« less

ORNLs Laboratory Directed Research and Development Program FY 2013 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

None, None

2014-03-01

The Laboratory Directed Research and Development (LDRD) program at Oak Ridge National Laboratory (ORNL) reports its status to the US Department of Energy (DOE) in March of each year. The program operates under the authority of DOE Order 413.2B, “Laboratory Directed Research and Development” (April 19, 2006), which establishes DOE’s requirements for the program while providing the Laboratory Director broad flexibility for program implementation. LDRD funds are obtained through a charge to all Laboratory programs. This report includes summaries of all ORNL LDRD research activities supported during FY 2013. The associated FY 2013 ORNL LDRD Self-Assessment (ORNL/PPA-2014/2) provides financial datamore » and an internal evaluation of the program’s management process.« less

ORNLs Laboratory Directed Research and Development Program FY 2008 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

None, None

2009-03-01

The Oak Ridge National Laboratory (ORNL) Laboratory Directed Research and Development (LDRD) Program reports its status to the U.S. Department of Energy (DOE) in March of each year. The program operates under the authority of DOE Order 413.2B, “Laboratory Directed Research and Development” (April 19, 2006), which establishes DOE’s requirements for the program while providing the Laboratory Director broad flexibility for program implementation. LDRD funds are obtained through a charge to all Laboratory programs. This report includes summaries all ORNL LDRD research activities supported during FY 2008. The associated FY 2008 ORNL LDRD Self-Assessment (ORNL/PPA-2008/2) provides financial data and anmore » internal evaluation of the program’s management process.« less

ORNLs Laboratory Directed Research and Development Program FY 2012 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

None, None

2013-03-01

The Laboratory Directed Research and Development (LDRD) program at Oak Ridge National Laboratory (ORNL) reports its status to the US Department of Energy (DOE) in March of each year. The program operates under the authority of DOE Order 413.2B, “Laboratory Directed Research and Development” (April 19, 2006), which establishes DOE’s requirements for the program while providing the Laboratory Director broad flexibility for program implementation. LDRD funds are obtained through a charge to all Laboratory programs. This report includes summaries of all ORNL LDRD research activities supported during FY 2012. The associated FY 2012 ORNL LDRD Self-Assessment (ORNL/PPA-2012/2) provides financial datamore » and an internal evaluation of the program’s management process.« less

A Nonstationary Markov Model Detects Directional Evolution in Hymenopteran Morphology.

PubMed

Klopfstein, Seraina; Vilhelmsen, Lars; Ronquist, Fredrik

2015-11-01

Directional evolution has played an important role in shaping the morphological, ecological, and molecular diversity of life. However, standard substitution models assume stationarity of the evolutionary process over the time scale examined, thus impeding the study of directionality. Here we explore a simple, nonstationary model of evolution for discrete data, which assumes that the state frequencies at the root differ from the equilibrium frequencies of the homogeneous evolutionary process along the rest of the tree (i.e., the process is nonstationary, nonreversible, but homogeneous). Within this framework, we develop a Bayesian approach for testing directional versus stationary evolution using a reversible-jump algorithm. Simulations show that when only data from extant taxa are available, the success in inferring directionality is strongly dependent on the evolutionary rate, the shape of the tree, the relative branch lengths, and the number of taxa. Given suitable evolutionary rates (0.1-0.5 expected substitutions between root and tips), accounting for directionality improves tree inference and often allows correct rooting of the tree without the use of an outgroup. As an empirical test, we apply our method to study directional evolution in hymenopteran morphology. We focus on three character systems: wing veins, muscles, and sclerites. We find strong support for a trend toward loss of wing veins and muscles, while stationarity cannot be ruled out for sclerites. Adding fossil and time information in a total-evidence dating approach, we show that accounting for directionality results in more precise estimates not only of the ancestral state at the root of the tree, but also of the divergence times. Our model relaxes the assumption of stationarity and reversibility by adding a minimum of additional parameters, and is thus well suited to studying the nature of the evolutionary process in data sets of limited size, such as morphology and ecology. © The Author

The Kjeldahl method as a primary reference procedure for total protein in certified reference materials used in clinical chemistry. II. Selection of direct Kjeldahl analysis and its preliminary performance parameters.

PubMed

Vinklárková, Bára; Chromý, Vratislav; Šprongl, Luděk; Bittová, Miroslava; Rikanová, Milena; Ohnútková, Ivana; Žaludová, Lenka

2015-01-01

To select a Kjeldahl procedure suitable for the determination of total protein in reference materials used in laboratory medicine, we reviewed in our previous article Kjeldahl methods adopted by clinical chemistry and found an indirect two-step analysis by total Kjeldahl nitrogen corrected for its nonprotein nitrogen and a direct analysis made on isolated protein precipitates. In this article, we compare both procedures on various reference materials. An indirect Kjeldahl method gave falsely lower results than a direct analysis. Preliminary performance parameters qualify the direct Kjeldahl analysis as a suitable primary reference procedure for the certification of total protein in reference laboratories.

Ultrafiltration of Protein Solutions: A Laboratory Experiment

ERIC Educational Resources Information Center

Pansare, Vikram J.; Tien, Daniel; Prud'homme, Robert K.

2015-01-01

Biology is playing an increasingly important role in the chemical engineering curriculum. We describe a set of experiments we have implemented in our Undergraduate Laboratory course giving students practical insights into membrane separation processes for protein processing. The goal of the lab is to optimize the purification and concentration of…

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

PubMed

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

2014-11-25

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

PubMed Central

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M.; Wong, Margaret; Kiessling, Laura L.; Steitz, Thomas A.; O’Donoghue, Patrick; Söll, Dieter

2014-01-01

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination

PubMed Central

Kim, Taejin; Chitteni-Pattu, Sindhu; Cox, Benjamin L.; Wood, Elizabeth A.; Sandler, Steven J.; Cox, Michael M.

2015-01-01

The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 recA gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism in vivo. The growth deficiency is alleviated by expression of the functionally robust RecX protein from Neisseria gonorrhoeae. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism. PMID:26047498

Protein change in plant evolution: tracing one thread connecting molecular and phenotypic diversity

PubMed Central

Bartlett, Madelaine E.; Whipple, Clinton J.

2013-01-01

Proteins change over the course of evolutionary time. New protein-coding genes and gene families emerge and diversify, ultimately affecting an organism’s phenotype and interactions with its environment. Here we survey the range of structural protein change observed in plants and review the role these changes have had in the evolution of plant form and function. Verified examples tying evolutionary change in protein structure to phenotypic change remain scarce. We will review the existing examples, as well as draw from investigations into domestication, and quantitative trait locus (QTL) cloning studies searching for the molecular underpinnings of natural variation. The evolutionary significance of many cloned QTL has not been assessed, but all the examples identified so far have begun to reveal the extent of protein structural diversity tolerated in natural systems. This molecular (and phenotypic) diversity could come to represent part of natural selection’s source material in the adaptive evolution of novel traits. Protein structure and function can change in many distinct ways, but the changes we identified in studies of natural diversity and protein evolution were predicted to fall primarily into one of six categories: altered active and binding sites; altered protein–protein interactions; altered domain content; altered activity as an activator or repressor; altered protein stability; and hypomorphic and hypermorphic alleles. There was also variability in the evolutionary scale at which particular changes were observed. Some changes were detected at both micro- and macroevolutionary timescales, while others were observed primarily at deep or shallow phylogenetic levels. This variation might be used to determine the trajectory of future investigations in structural molecular evolution. PMID:24124420

Selection platforms for directed evolution in synthetic biology.

PubMed

Tizei, Pedro A G; Csibra, Eszter; Torres, Leticia; Pinheiro, Vitor B

2016-08-15

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules-gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function-be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code. © 2016 The Author(s).

Laboratory Directed Research and Development Program Activities for FY 2007.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Newman,L.

2007-12-31

Brookhaven National Laboratory (BNL) is a multidisciplinary laboratory that carries out basic and applied research in the physical, biomedical, and environmental sciences, and in selected energy technologies. It is managed by Brookhaven Science Associates, LLC, (BSA) under contract with the U. S. Department of Energy (DOE). BNL's Fiscal year 2007 budget was $515 million. There are about 2,600 employees, and another 4,500 guest scientists and students who come each year to use the Laboratory's facilities and work with the staff. The BNL Laboratory Directed Research and Development (LDRD) Program reports its status to the U.S. Department of Energy (DOE) annuallymore » in March, as required by DOE Order 413.2B, 'Laboratory Directed Research and Development', April 19, 2006, and the Roles, Responsibilities, and Guidelines for Laboratory Directed Research and Development at the Department of Energy/National Nuclear Security Administration Laboratories dated June 13, 2006. In accordance this is our Annual Report in which we describe the Purpose, Approach, Technical Progress and Results, and Specific Accomplishments of all LDRD projects that received funding during Fiscal Year 2007. The goals and objectives of BNL's LDRD Program can be inferred from the Program's stated purposes. These are to (1) encourage and support the development of new ideas and technology, (2) promote the early exploration and exploitation of creative and innovative concepts, and (3) develop new 'fundable' R&D projects and programs. The emphasis is clearly articulated by BNL to be on supporting exploratory research 'which could lead to new programs, projects, and directions' for the Laboratory. We explicitly indicate that research conducted under the LDRD Program should be highly innovative, and an element of high risk as to success is acceptable. In the solicitation for new proposals for Fiscal Year 2007 we especially requested innovative new projects in support of RHIC and the Light Source and

Anomalous diffusion in neutral evolution of model proteins.

PubMed

Nelson, Erik D; Grishin, Nick V

2015-06-01

Protein evolution is frequently explored using minimalist polymer models, however, little attention has been given to the problem of structural drift, or diffusion. Here, we study neutral evolution of small protein motifs using an off-lattice heteropolymer model in which individual monomers interact as low-resolution amino acids. In contrast to most earlier models, both the length and folded structure of the polymers are permitted to change. To describe structural change, we compute the mean-square distance (MSD) between monomers in homologous folds separated by n neutral mutations. We find that structural change is episodic, and, averaged over lineages (for example, those extending from a single sequence), exhibits a power-law dependence on n. We show that this exponent depends on the alignment method used, and we analyze the distribution of waiting times between neutral mutations. The latter are more disperse than for models required to maintain a specific fold, but exhibit a similar power-law tail.

Anomalous diffusion in neutral evolution of model proteins

NASA Astrophysics Data System (ADS)

Nelson, Erik D.; Grishin, Nick V.

2015-06-01

Protein evolution is frequently explored using minimalist polymer models, however, little attention has been given to the problem of structural drift, or diffusion. Here, we study neutral evolution of small protein motifs using an off-lattice heteropolymer model in which individual monomers interact as low-resolution amino acids. In contrast to most earlier models, both the length and folded structure of the polymers are permitted to change. To describe structural change, we compute the mean-square distance (MSD) between monomers in homologous folds separated by n neutral mutations. We find that structural change is episodic, and, averaged over lineages (for example, those extending from a single sequence), exhibits a power-law dependence on n . We show that this exponent depends on the alignment method used, and we analyze the distribution of waiting times between neutral mutations. The latter are more disperse than for models required to maintain a specific fold, but exhibit a similar power-law tail.

Protein Secretion Systems in Pseudomonas aeruginosa: An Essay on Diversity, Evolution, and Function

PubMed Central

Filloux, Alain

2011-01-01

Protein secretion systems are molecular nanomachines used by Gram-negative bacteria to thrive within their environment. They are used to release enzymes that hydrolyze complex carbon sources into usable compounds, or to release proteins that capture essential ions such as iron. They are also used to colonize and survive within eukaryotic hosts, causing acute or chronic infections, subverting the host cell response and escaping the immune system. In this article, the opportunistic human pathogen Pseudomonas aeruginosa is used as a model to review the diversity of secretion systems that bacteria have evolved to achieve these goals. This diversity may result from a progressive transformation of cell envelope complexes that initially may not have been dedicated to secretion. The striking similarities between secretion systems and type IV pili, flagella, bacteriophage tail, or efflux pumps is a nice illustration of this evolution. Differences are also needed since various secretion configurations call for diversity. For example, some proteins are released in the extracellular medium while others are directly injected into the cytosol of eukaryotic cells. Some proteins are folded before being released and transit into the periplasm. Other proteins cross the whole cell envelope at once in an unfolded state. However, the secretion system requires conserved basic elements or features. For example, there is a need for an energy source or for an outer membrane channel. The structure of this review is thus quite unconventional. Instead of listing secretion types one after each other, it presents a melting pot of concepts indicating that secretion types are in constant evolution and use basic principles. In other words, emergence of new secretion systems could be predicted the way Mendeleïev had anticipated characteristics of yet unknown elements. PMID:21811488

Benefits of a Recombination-Proficient Escherichia coli System for Adaptive Laboratory Evolution.

PubMed

Peabody, George; Winkler, James; Fountain, Weston; Castro, David A; Leiva-Aravena, Enzo; Kao, Katy C

2016-11-15

Adaptive laboratory evolution typically involves the propagation of organisms asexually to select for mutants with the desired phenotypes. However, asexual evolution is prone to competition among beneficial mutations (clonal interference) and the accumulation of hitchhiking and neutral mutations. The benefits of horizontal gene transfer toward overcoming these known disadvantages of asexual evolution were characterized in a strain of Escherichia coli engineered for superior sexual recombination (genderless). Specifically, we experimentally validated the capacity of the genderless strain to reduce the mutational load and recombine beneficial mutations. We also confirmed that inclusion of multiple origins of transfer influences both the frequency of genetic exchange throughout the chromosome and the linkage of donor DNA. We built a simple kinetic model to estimate recombination frequency as a function of transfer size and relative genotype enrichment in batch transfers; the model output correlated well with the experimental data. Our results provide strong support for the advantages of utilizing the genderless strain over its asexual counterpart during adaptive laboratory evolution for generating beneficial mutants with reduced mutational load. Over 80 years ago Fisher and Muller began a debate on the origins of sexual recombination. Although many aspects of sexual recombination have been examined at length, experimental evidence behind the behaviors of recombination in many systems and the means to harness it remain elusive. In this study, we sought to experimentally validate some advantages of recombination in typically asexual Escherichia coli and determine if a sexual strain of E. coli can become an effective tool for strain development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Benefits of a Recombination-Proficient Escherichia coli System for Adaptive Laboratory Evolution

PubMed Central

Peabody, George; Winkler, James; Fountain, Weston; Castro, David A.; Leiva-Aravena, Enzo

2016-01-01

ABSTRACT Adaptive laboratory evolution typically involves the propagation of organisms asexually to select for mutants with the desired phenotypes. However, asexual evolution is prone to competition among beneficial mutations (clonal interference) and the accumulation of hitchhiking and neutral mutations. The benefits of horizontal gene transfer toward overcoming these known disadvantages of asexual evolution were characterized in a strain of Escherichia coli engineered for superior sexual recombination (genderless). Specifically, we experimentally validated the capacity of the genderless strain to reduce the mutational load and recombine beneficial mutations. We also confirmed that inclusion of multiple origins of transfer influences both the frequency of genetic exchange throughout the chromosome and the linkage of donor DNA. We built a simple kinetic model to estimate recombination frequency as a function of transfer size and relative genotype enrichment in batch transfers; the model output correlated well with the experimental data. Our results provide strong support for the advantages of utilizing the genderless strain over its asexual counterpart during adaptive laboratory evolution for generating beneficial mutants with reduced mutational load. IMPORTANCE Over 80 years ago Fisher and Muller began a debate on the origins of sexual recombination. Although many aspects of sexual recombination have been examined at length, experimental evidence behind the behaviors of recombination in many systems and the means to harness it remain elusive. In this study, we sought to experimentally validate some advantages of recombination in typically asexual Escherichia coli and determine if a sexual strain of E. coli can become an effective tool for strain development. PMID:27613685

Laboratory Directed Research and Development Program Assessment for FY 2016

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatton, Diane; Flynn, Liz

2017-03-31

Each year, Brookhaven National Laboratory (BNL) is required to provide a program description and overview of its Laboratory Directed Research and Development Program (LDRD) to the Department of Energy in accordance with DOE Order 413.2C, and this report fulfills that requirement.

Laboratory Directed Research and Development Program Assessment for FY 2017

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, Jack; Flynn, Liz

Each year, Brookhaven National Laboratory (BNL) is required to provide a program description and overview of its Laboratory Directed Research and Development Program (LDRD) to the Department of Energy in accordance with DOE Order 413.2C. This report fulfills that requirement.

Exploring protein structure and dynamics through a project-oriented biochemistry laboratory module.

PubMed

Lipchock, James M; Ginther, Patrick S; Douglas, Bonnie B; Bird, Kelly E; Patrick Loria, J

2017-09-01

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics. This project-based approach allows an instructor to simulate a research setting and prepare students for productive research beyond the classroom. Potential modifications to expand or contract this module are also provided. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):403-410, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Evolution dependence of vanadium nitride nanoprecipitates on directionality of ion irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, L.; Kim, B. K.; Was, G. S.

The influence of the directionality of Fe 2+ ion irradiation on the evolution of vanadium nitride platelet–shaped nanoprecipitates at 500 °C was investigated in this paper in a ferritic alloy using transmission electron microscopy. When the ion-irradiation direction was approximately aligned with the initial particle length, particles grew longer and sectioned into shorter lengths at higher doses, resulting in increased particle densities. As ion-irradiation direction deviated from particle-length direction, some particles sectioned lengthwise and then dissolved, resulting in decreased particle densities. Surviving particles were transformed into parallelograms with a different orientation relationship with the matrix. Finally, nanoprecipitate evolution dependence onmore » beam-nanoprecipitate orientation is a process that may be different from reactor irradiation.« less

Evolution dependence of vanadium nitride nanoprecipitates on directionality of ion irradiation

DOE PAGES

Tan, L.; Kim, B. K.; Was, G. S.

2017-09-06

The influence of the directionality of Fe 2+ ion irradiation on the evolution of vanadium nitride platelet–shaped nanoprecipitates at 500 °C was investigated in this paper in a ferritic alloy using transmission electron microscopy. When the ion-irradiation direction was approximately aligned with the initial particle length, particles grew longer and sectioned into shorter lengths at higher doses, resulting in increased particle densities. As ion-irradiation direction deviated from particle-length direction, some particles sectioned lengthwise and then dissolved, resulting in decreased particle densities. Surviving particles were transformed into parallelograms with a different orientation relationship with the matrix. Finally, nanoprecipitate evolution dependence onmore » beam-nanoprecipitate orientation is a process that may be different from reactor irradiation.« less

Protein interactions and ligand binding: from protein subfamilies to functional specificity.

PubMed

Rausell, Antonio; Juan, David; Pazos, Florencio; Valencia, Alfonso

2010-02-02

The divergence accumulated during the evolution of protein families translates into their internal organization as subfamilies, and it is directly reflected in the characteristic patterns of differentially conserved residues. These specifically conserved positions in protein subfamilies are known as "specificity determining positions" (SDPs). Previous studies have limited their analysis to the study of the relationship between these positions and ligand-binding specificity, demonstrating significant yet limited predictive capacity. We have systematically extended this observation to include the role of differential protein interactions in the segregation of protein subfamilies and explored in detail the structural distribution of SDPs at protein interfaces. Our results show the extensive influence of protein interactions in the evolution of protein families and the widespread association of SDPs with protein interfaces. The combined analysis of SDPs in interfaces and ligand-binding sites provides a more complete picture of the organization of protein families, constituting the necessary framework for a large scale analysis of the evolution of protein function.

Comparable contributions of structural-functional constraints and expression level to the rate of protein sequence evolution

PubMed Central

Wolf, Maxim Y; Wolf, Yuri I; Koonin, Eugene V

2008-01-01

Background Proteins show a broad range of evolutionary rates. Understanding the factors that are responsible for the characteristic rate of evolution of a given protein arguably is one of the major goals of evolutionary biology. A long-standing general assumption used to be that the evolution rate is, primarily, determined by the specific functional constraints that affect the given protein. These constrains were traditionally thought to depend both on the specific features of the protein's structure and its biological role. The advent of systems biology brought about new types of data, such as expression level and protein-protein interactions, and unexpectedly, a variety of correlations between protein evolution rate and these variables have been observed. The strongest connections by far were repeatedly seen between protein sequence evolution rate and the expression level of the respective gene. It has been hypothesized that this link is due to the selection for the robustness of the protein structure to mistranslation-induced misfolding that is particularly important for highly expressed proteins and is the dominant determinant of the sequence evolution rate. Results This work is an attempt to assess the relative contributions of protein domain structure and function, on the one hand, and expression level on the other hand, to the rate of sequence evolution. To this end, we performed a genome-wide analysis of the effect of the fusion of a pair of domains in multidomain proteins on the difference in the domain-specific evolutionary rates. The mistranslation-induced misfolding hypothesis would predict that, within multidomain proteins, fused domains, on average, should evolve at substantially closer rates than the same domains in different proteins because, within a mutlidomain protein, all domains are translated at the same rate. We performed a comprehensive comparison of the evolutionary rates of mammalian and plant protein domains that are either joined in

Protein evolution analysis of S-hydroxynitrile lyase by complete sequence design utilizing the INTMSAlign software.

PubMed

Nakano, Shogo; Asano, Yasuhisa

2015-02-03

Development of software and methods for design of complete sequences of functional proteins could contribute to studies of protein engineering and protein evolution. To this end, we developed the INTMSAlign software, and used it to design functional proteins and evaluate their usefulness. The software could assign both consensus and correlation residues of target proteins. We generated three protein sequences with S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNLs; these proteins folded as efficiently as the native S-HNL. Sequence and biochemical analysis of the designed S-HNLs suggested that accumulation of neutral mutations occurs during the process of S-HNLs evolution from a low-activity form to a high-activity (native) form. Taken together, our results demonstrate that our software and the associated methods could be applied not only to design of complete sequences, but also to predictions of protein evolution, especially within families such as esterases and S-HNLs.

Protein evolution analysis of S-hydroxynitrile lyase by complete sequence design utilizing the INTMSAlign software

NASA Astrophysics Data System (ADS)

Nakano, Shogo; Asano, Yasuhisa

2015-02-01

Development of software and methods for design of complete sequences of functional proteins could contribute to studies of protein engineering and protein evolution. To this end, we developed the INTMSAlign software, and used it to design functional proteins and evaluate their usefulness. The software could assign both consensus and correlation residues of target proteins. We generated three protein sequences with S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNLs; these proteins folded as efficiently as the native S-HNL. Sequence and biochemical analysis of the designed S-HNLs suggested that accumulation of neutral mutations occurs during the process of S-HNLs evolution from a low-activity form to a high-activity (native) form. Taken together, our results demonstrate that our software and the associated methods could be applied not only to design of complete sequences, but also to predictions of protein evolution, especially within families such as esterases and S-HNLs.

Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis

PubMed Central

2012-01-01

Background Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes. Results Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory. Conclusions Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types. PMID:22873208

Laboratory Directed Research and Development FY-10 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dena Tomchak

2011-03-01

The FY 2010 Laboratory Directed Research and Development (LDRD) Annual Report is a compendium of the diverse research performed to develop and ensure the INL's technical capabilities can support the future DOE missions and national research priorities. LDRD is essential to the INL -- it provides a means for the laboratory to pursue novel scientific and engineering research in areas that are deemed too basic or risky for programmatic investments. This research enhances technical capabilities at the laboratory, providing scientific and engineering staff with opportunities for skill building and partnership development.

Maternal Lipid Provisioning Mirrors Evolution of Reproductive Strategies in Direct-Developing Whelks.

PubMed

Carrasco, Sergio A; Phillips, Nicole E; Sewell, Mary A

2016-06-01

The energetic input that offspring receive from their mothers is a well-studied maternal effect that can influence the evolution of life histories. Using the offspring of three sympatric whelks: Cominella virgata (one embryo per capsule); Cominella maculosa (multiple embryos per capsule); and Haustrum scobina (multiple embryos per capsule and nurse-embryo consumption), we examined how contrasting reproductive strategies mediate inter- and intraspecific differences in hatchling provisioning. Total lipid content (as measured in μg hatchling(-1) ± SE) was unrelated to size among the 3 species; the hatchlings of H. scobina were the smallest but had the highest lipid content (33.8 ± 8.1 μg hatchling(-1)). In offspring of C. maculosa, lipid content was 6.6 ± 0.4 μg hatchling(-1), and in offspring of C. virgata, it was 21.7 ± 3.2 μg hatchling(-1) The multi-encapsulated hatchlings of C. maculosa and H. scobina were the only species that contained the energetic lipids, wax ester (WE) and methyl ester (ME). However, the overall composition of energetic lipid between hatchlings of the two Cominella species reflected strong affinities of taxonomy, suggesting a phylogenetic evolution of the non-adelphophagic development strategy. Inter- and intracapsular variability in sibling provisioning was highest in H. scobina, a finding that implies less control of allocation to individual hatchlings in this adelphophagic developer. We suggest that interspecific variability of lipids offers a useful approach to understanding the evolution of maternal provisioning in direct-developing species. © 2016 Marine Biological Laboratory.

Tempo and Mode of Gene Duplication in Mammalian Ribosomal Protein Evolution

PubMed Central

Gajdosik, Matthew D.; Simon, Amanda; Nelson, Craig E.

2014-01-01

Gene duplication has been widely recognized as a major driver of evolutionary change and organismal complexity through the generation of multi-gene families. Therefore, understanding the forces that govern the evolution of gene families through the retention or loss of duplicated genes is fundamentally important in our efforts to study genome evolution. Previous work from our lab has shown that ribosomal protein (RP) genes constitute one of the largest classes of conserved duplicated genes in mammals. This result was surprising due to the fact that ribosomal protein genes evolve slowly and transcript levels are very tightly regulated. In our present study, we identified and characterized all RP duplicates in eight mammalian genomes in order to investigate the tempo and mode of ribosomal protein family evolution. We show that a sizable number of duplicates are transcriptionally active and are very highly conserved. Furthermore, we conclude that existing gene duplication models do not readily account for the preservation of a very large number of intact retroduplicated ribosomal protein (RT-RP) genes observed in mammalian genomes. We suggest that selection against dominant-negative mutations may underlie the unexpected retention and conservation of duplicated RP genes, and may shape the fate of newly duplicated genes, regardless of duplication mechanism. PMID:25369106

Co-evolution of SNF spliceosomal proteins with their RNA targets in trans-splicing nematodes.

PubMed

Strange, Rex Meade; Russelburg, L Peyton; Delaney, Kimberly J

2016-08-01

Although the mechanism of pre-mRNA splicing has been well characterized, the evolution of spliceosomal proteins is poorly understood. The U1A/U2B″/SNF family (hereafter referred to as the SNF family) of RNA binding spliceosomal proteins participates in both the U1 and U2 small interacting nuclear ribonucleoproteins (snRNPs). The highly constrained nature of this system has inhibited an analysis of co-evolutionary trends between the proteins and their RNA binding targets. Here we report accelerated sequence evolution in the SNF protein family in Phylum Nematoda, which has allowed an analysis of protein:RNA co-evolution. In a comparison of SNF genes from ecdysozoan species, we found a correlation between trans-splicing species (nematodes) and increased phylogenetic branch lengths of the SNF protein family, with respect to their sister clade Arthropoda. In particular, we found that nematodes (~70-80 % of pre-mRNAs are trans-spliced) have experienced higher rates of SNF sequence evolution than arthropods (predominantly cis-spliced) at both the nucleotide and amino acid levels. Interestingly, this increased evolutionary rate correlates with the reliance on trans-splicing by nematodes, which would alter the role of the SNF family of spliceosomal proteins. We mapped amino acid substitutions to functionally important regions of the SNF protein, specifically to sites that are predicted to disrupt protein:RNA and protein:protein interactions. Finally, we investigated SNF's RNA targets: the U1 and U2 snRNAs. Both are more divergent in nematodes than arthropods, suggesting the RNAs have co-evolved with SNF in order to maintain the necessarily high affinity interaction that has been characterized in other species.

Laboratory Directed Research and Development Program Assessment for FY 2008

DOE Office of Scientific and Technical Information (OSTI.GOV)

Looney, J P; Fox, K J

2008-03-31

Brookhaven National Laboratory (BNL) is a multidisciplinary Laboratory that carries out basic and applied research in the physical, biomedical, and environmental sciences, and in selected energy technologies. It is managed by Brookhaven Science Associates, LLC, (BSA) under contract with the U. S. Department of Energy (DOE). BNL's Fiscal Year 2008 spending was $531.6 million. There are approximately 2,800 employees, and another 4,300 guest scientists and students who come each year to use the Laboratory's facilities and work with the staff. The BNL Laboratory Directed Research and Development (LDRD) Program reports its status to the U.S. Department of Energy (DOE) annuallymore » in March, as required by DOE Order 413.2B, 'Laboratory Directed Research and Development,' April 19, 2006, and the Roles, Responsibilities, and Guidelines for Laboratory Directed Research and Development at the Department of Energy/National Nuclear Security Administration Laboratories dated June 13, 2006. The goals and objectives of BNL's LDRD Program can be inferred from the Program's stated purposes. These are to (1) encourage and support the development of new ideas and technology, (2) promote the early exploration and exploitation of creative and innovative concepts, and (3) develop new 'fundable' R&D projects and programs. The emphasis is clearly articulated by BNL to be on supporting exploratory research 'which could lead to new programs, projects, and directions' for the Laboratory. To be a premier scientific Laboratory, BNL must continuously foster groundbreaking scientific research and renew its research agenda. The competition for LDRD funds stimulates Laboratory scientists to think in new and creative ways, which becomes a major factor in achieving and maintaining research excellence and a means to address National needs within the overall mission of the DOE and BNL. By fostering high-risk, exploratory research, the LDRD program helps BNL to respond new scientific opportunities

Models of Protocellular Structure, Function and Evolution

NASA Technical Reports Server (NTRS)

New, Michael H.; Pohorille, Andrew; Szostak, Jack W.; Keefe, Tony; Lanyi, Janos K.

2001-01-01

In the absence of any record of protocells, the most direct way to test our understanding of the origin of cellular life is to construct laboratory models that capture important features of protocellular systems. Such efforts are currently underway in a collaborative project between NASA-Ames, Harvard Medical School and University of California. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures. The centerpiece of this project is a method for the in vitro evolution of protein enzymes toward arbitrary catalytic targets. A similar approach has already been developed for nucleic acids in which a small number of functional molecules are selected from a large, random population of candidates. The selected molecules are next vastly multiplied using the polymerase chain reaction. A mutagenic approach, in which the sequences of selected molecules are randomly altered, can yield further improvements in performance or alterations of specificities. Unfortunately, the catalytic potential of nucleic acids is rather limited. Proteins are more catalytically capable but cannot be directly amplified. In the new technique, this problem is circumvented by covalently linking each protein of the initial, diverse, pool to the RNA sequence that codes for it. Then, selection is performed on the proteins, but the nucleic acids are replicated. Additional information is contained in the original extended abstract.

Laboratory directed research and development 2006 annual report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Westrich, Henry Roger

2007-03-01

This report summarizes progress from the Laboratory Directed Research and Development (LDRD) program during fiscal year 2006. In addition to a programmatic and financial overview, the report includes progress reports from 430 individual R&D projects in 17 categories.

Dwarf Galaxies: Laboratories for Nucleosynthesis and Chemical Evolution

NASA Astrophysics Data System (ADS)

Kirby, Evan N.

2018-06-01

The dwarf galaxies in the Local Group are excellent laboratories for studying the creation of the elements (nucleosynthesis) and the build-up of those elements over time (chemical evolution). The galaxies' proximity permits spectroscopy of individual stars, from which detailed elemental abundances can be measured. Their small sizes and, in some cases, short star formation lifetimes imprinted chemical histories that are easy to interpret relative to larger, more complex galaxies, like the Milky Way.I will briefly review some techniques for measuring elemental abundances from medium-resolution spectroscopy of individual stars. I will show how the metallicity distributions of dwarf galaxies reflect their gas content at the time they were forming stars. Then, I will show how the ratio of alpha elements (for example, magnesium) to iron reveals the star formation history. Finally, I will use certain elements to tease out details of nucleosynthetic events. For example, low manganese and cobalt abundances indicate that the typical Type Ia supernova in dwarf galaxies was a low-density white dwarf, and the evolution of barium suggests that neutron star mergers were most likely responsible for the majority of neutron-capture elements in smaller dwarf galaxies.

From Green to Blue: Site-Directed Mutagenesis of the Green Fluorescent Protein to Teach Protein Structure-Function Relationships

ERIC Educational Resources Information Center

Giron, Maria D.; Salto, Rafael

2011-01-01

Structure-function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the…

Principal Component Analysis reveals correlation of cavities evolution and functional motions in proteins.

PubMed

Desdouits, Nathan; Nilges, Michael; Blondel, Arnaud

2015-02-01

Protein conformation has been recognized as the key feature determining biological function, as it determines the position of the essential groups specifically interacting with substrates. Hence, the shape of the cavities or grooves at the protein surface appears to drive those functions. However, only a few studies describe the geometrical evolution of protein cavities during molecular dynamics simulations (MD), usually with a crude representation. To unveil the dynamics of cavity geometry evolution, we developed an approach combining cavity detection and Principal Component Analysis (PCA). This approach was applied to four systems subjected to MD (lysozyme, sperm whale myoglobin, Dengue envelope protein and EF-CaM complex). PCA on cavities allows us to perform efficient analysis and classification of the geometry diversity explored by a cavity. Additionally, it reveals correlations between the evolutions of the cavities and structures, and can even suggest how to modify the protein conformation to induce a given cavity geometry. It also helps to perform fast and consensual clustering of conformations according to cavity geometry. Finally, using this approach, we show that both carbon monoxide (CO) location and transfer among the different xenon sites of myoglobin are correlated with few cavity evolution modes of high amplitude. This correlation illustrates the link between ligand diffusion and the dynamic network of internal cavities. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Trends in the sand: Directional evolution in the shell shape of recessing scallops (Bivalvia: Pectinidae).

PubMed

Sherratt, Emma; Alejandrino, Alvin; Kraemer, Andrew C; Serb, Jeanne M; Adams, Dean C

2016-09-01

Directional evolution is one of the most compelling evolutionary patterns observed in macroevolution. Yet, despite its importance, detecting such trends in multivariate data remains a challenge. In this study, we evaluate multivariate evolution of shell shape in 93 bivalved scallop species, combining geometric morphometrics and phylogenetic comparative methods. Phylomorphospace visualization described the history of morphological diversification in the group; revealing that taxa with a recessing life habit were the most distinctive in shell shape, and appeared to display a directional trend. To evaluate this hypothesis empirically, we extended existing methods by characterizing the mean directional evolution in phylomorphospace for recessing scallops. We then compared this pattern to what was expected under several alternative evolutionary scenarios using phylogenetic simulations. The observed pattern did not fall within the distribution obtained under multivariate Brownian motion, enabling us to reject this evolutionary scenario. By contrast, the observed pattern was more similar to, and fell within, the distribution obtained from simulations using Brownian motion combined with a directional trend. Thus, the observed data are consistent with a pattern of directional evolution for this lineage of recessing scallops. We discuss this putative directional evolutionary trend in terms of its potential adaptive role in exploiting novel habitats. © 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.

Direct Detection of Biotinylated Proteins by Mass Spectrometry

PubMed Central

2015-01-01

Mass spectrometric strategies to identify protein subpopulations involved in specific biological functions rely on covalently tagging biotin to proteins using various chemical modification methods. The biotin tag is primarily used for enrichment of the targeted subpopulation for subsequent mass spectrometry (MS) analysis. A limitation of these strategies is that MS analysis does not easily discriminate unlabeled contaminants from the labeled protein subpopulation under study. To solve this problem, we developed a flexible method that only relies on direct MS detection of biotin-tagged proteins called “Direct Detection of Biotin-containing Tags” (DiDBiT). Compared with conventional targeted proteomic strategies, DiDBiT improves direct detection of biotinylated proteins ∼200 fold. We show that DiDBiT is applicable to several protein labeling protocols in cell culture and in vivo using cell permeable NHS-biotin and incorporation of the noncanonical amino acid, azidohomoalanine (AHA), into newly synthesized proteins, followed by click chemistry tagging with biotin. We demonstrate that DiDBiT improves the direct detection of biotin-tagged newly synthesized peptides more than 20-fold compared to conventional methods. With the increased sensitivity afforded by DiDBiT, we demonstrate the MS detection of newly synthesized proteins labeled in vivo in the rodent nervous system with unprecedented temporal resolution as short as 3 h. PMID:25117199

Manipulating heat shock protein expression in laboratory animals.

PubMed

Tolson, J Keith; Roberts, Stephen M

2005-02-01

Upregulation of heat shock proteins (Hsps) has been observed to impart resistance to a wide variety of physical and chemical insults. Elucidation of the role of Hsps in cellular defense processes depends, in part, on the ability to manipulate Hsp expression in laboratory animals. Simple methods of inducing whole body hyperthermia, such as warm water immersion or heating pad application, are effective in producing generalized expression of Hsps. Hsps can be upregulated locally with focused direct or indirect heating, such as with ultrasound or with laser or microwave radiation. Increased Hsp expression in response to toxic doses of xenobiotics has been commonly observed. Some pharmacologic agents are capable of altering Hsps more specifically by affecting processes involved in Hsp regulation. Gene manipulation offers the ability to selectively increase or decrease individual Hsps. Knockout mouse strains and Hsp-overexpressing transgenics have been used successfully to examine the role of specific Hsps in protection against hyperthermia, chemical insults, and ischemia-reperfusion injury. Gene therapy approaches also offer the possibility of selective alteration of Hsp expression. Some methods of increasing Hsp expression have application in specialized areas of research, such cold response, myocardial protection from exercise, and responses to stressful or traumatic stimuli. Each method of manipulating Hsp expression in laboratory animals has advantages and disadvantages, and selection of the best method depends upon the experimental objectives (e.g., the alteration in Hsp expression needed, its timing, and its location) and resources available.

Oligovalent Fab display on M13 phage improved by directed evolution.

PubMed

Huovinen, Tuomas; Sanmark, Hanna; Ylä-Pelto, Jani; Vehniäinen, Markus; Lamminmäki, Urpo

2010-03-01

Efficient display of antibody on filamentous phage M13 coat is crucial for successful biopanning selections. We applied a directed evolution strategy to improve the oligovalent display of a poorly behaving Fab fragment fused to phage gene-3 for minor coat protein (g3p). The Fab displaying clones were enriched from a randomly mutated Fab gene library with polyclonal anti-mouse IgG antibodies. Contribution of each mutation to the improved phenotype of one selected mutant was studied. It was found out that two point mutations had significant contribution to the display efficiency of Fab clones superinfected with hyperphage. The most dramatic effect was connected to a start codon mutation, from AUG to GUG, of the PelB signal sequence preceding the heavy chain. The clone carrying this mutation, FabM(GUG), displayed Fab 19-fold better and yielded twofold higher phage titers than the original Fab.

Evolution of an ancient protein function involved in organized multicellularity in animals.

PubMed

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-07

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which - the evolution of GKPID's capacity to bind the cortical marker protein - can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals.

2014 Fermilab Laboratory Directed Research & Development Program Plan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wester, W., editor

2016-05-26

Fermilab is executing Laboratory Directed Research and Development (LDRD) as outlined by order DOE O 413.2B in order to enhance and realize the mission of the laboratory in a manner that also supports the laboratory’s strategic objectives and the mission of the Department of Energy. LDRD funds enable scientific creativity, allow for exploration of “high risk, high payoff” research, and allow for the demonstration of new ideas, technical concepts, and devices. LDRD also has an objective of maintaining and enhancing the scientific and technical vitality of Fermilab.

Teaching Noncovalent Interactions Using Protein Molecular Evolution

ERIC Educational Resources Information Center

Fornasari, Maria Silvina; Parisi, Gustavo; Echave, Julian

2008-01-01

Noncovalent interactions and physicochemical properties of amino acids are important topics in biochemistry courses. Here, we present a computational laboratory where the capacity of each of the 20 amino acids to maintain different noncovalent interactions are used to investigate the stabilizing forces in a set of proteins coming from organisms…

Laboratory Directed Research and Development annual report, fiscal year 1997

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1998-03-01

The Department of Energy Order 413.2(a) establishes DOE`s policy and guidelines regarding Laboratory Directed Research and Development (LDRD) at its multiprogram laboratories. As described in 413.2, LDRD is research and development of a creative and innovative nature which is selected by the Laboratory Director or his or her designee, for the purpose of maintaining the scientific and technological vitality of the Laboratory and to respond to scientific and technological opportunities in conformance with the guidelines in this Order. DOE Order 413.2 requires that each laboratory submit an annual report on its LDRD activities to the cognizant Secretarial Officer through themore » appropriate Operations Office Manager. The report provided in this document represents Pacific Northwest National Laboratory`s LDRD report for FY 1997.« less

A practical teaching course in directed protein evolution using the green fluorescent protein as a model.

PubMed

Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Cruz Schneider, Maria Paula; Ward, Richard John

2011-01-01

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility. Copyright © 2011 Wiley Periodicals, Inc.

Detecting Selection on Protein Stability through Statistical Mechanical Models of Folding and Evolution

PubMed Central

Bastolla, Ugo

2014-01-01

The properties of biomolecules depend both on physics and on the evolutionary process that formed them. These two points of view produce a powerful synergism. Physics sets the stage and the constraints that molecular evolution has to obey, and evolutionary theory helps in rationalizing the physical properties of biomolecules, including protein folding thermodynamics. To complete the parallelism, protein thermodynamics is founded on the statistical mechanics in the space of protein structures, and molecular evolution can be viewed as statistical mechanics in the space of protein sequences. In this review, we will integrate both points of view, applying them to detecting selection on the stability of the folded state of proteins. We will start discussing positive design, which strengthens the stability of the folded against the unfolded state of proteins. Positive design justifies why statistical potentials for protein folding can be obtained from the frequencies of structural motifs. Stability against unfolding is easier to achieve for longer proteins. On the contrary, negative design, which consists in destabilizing frequently formed misfolded conformations, is more difficult to achieve for longer proteins. The folding rate can be enhanced by strengthening short-range native interactions, but this requirement contrasts with negative design, and evolution has to trade-off between them. Finally, selection can accelerate functional movements by favoring low frequency normal modes of the dynamics of the native state that strongly correlate with the functional conformation change. PMID:24970217

2015 Fermilab Laboratory Directed Research & Development Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wester, W.

2016-05-26

The Fermi National Accelerator Laboratory (FNAL) is conducting a Laboratory Directed Research and Development (LDRD) program. Fiscal year 2015 represents the first full year of LDRD at Fermilab and includes seven projects approved mid-year in FY14 and six projects approved in FY15. One of the seven original projects has been completed just after the beginning of FY15. The implementation of LDRD at Fermilab is captured in the approved Fermilab 2015 LDRD Annual Program Plan. In FY15, the LDRD program represents 0.64% of Laboratory funding. The scope of the LDRD program at Fermilab will be established over the next couple ofmore » years where a portfolio of about 20 on-going projects representing approximately between 1% and 1.5% of the Laboratory funding is anticipated. This Annual Report focuses on the status of the current projects and provides an overview of the current status of LDRD at Fermilab.« less

Positive Selection Drives the Evolution of rhino, a Member of the Heterochromatin Protein 1 Family in Drosophila

PubMed Central

Vermaak, Danielle; Henikoff, Steven; Malik, Harmit S

2005-01-01

Heterochromatin comprises a significant component of many eukaryotic genomes. In comparison to euchromatin, heterochromatin is gene poor, transposon rich, and late replicating. It serves many important biological roles, from gene silencing to accurate chromosome segregation, yet little is known about the evolutionary constraints that shape heterochromatin. A complementary approach to the traditional one of directly studying heterochromatic DNA sequence is to study the evolution of proteins that bind and define heterochromatin. One of the best markers for heterochromatin is the heterochromatin protein 1 (HP1), which is an essential, nonhistone chromosomal protein. Here we investigate the molecular evolution of five HP1 paralogs present in Drosophila melanogaster. Three of these paralogs have ubiquitous expression patterns in adult Drosophila tissues, whereas HP1D/rhino and HP1E are expressed predominantly in ovaries and testes respectively. The HP1 paralogs also have distinct localization preferences in Drosophila cells. Thus, Rhino localizes to the heterochromatic compartment in Drosophila tissue culture cells, but in a pattern distinct from HP1A and lysine-9 dimethylated H3. Using molecular evolution and population genetic analyses, we find that rhino has been subject to positive selection in all three domains of the protein: the N-terminal chromo domain, the C-terminal chromo-shadow domain, and the hinge region that connects these two modules. Maximum likelihood analysis of rhino sequences from 20 species of Drosophila reveals that a small number of residues of the chromo and shadow domains have been subject to repeated positive selection. The rapid and positive selection of rhino is highly unusual for a gene encoding a chromosomal protein and suggests that rhino is involved in a genetic conflict that affects the germline, belying the notion that heterochromatin is simply a passive recipient of “junk DNA” in eukaryotic genomes. PMID:16103923

Different Cells Make Different Proteins: A Laboratory Exercise Illustrating Tissue-Specific Protein Expression in Animals

ERIC Educational Resources Information Center

Ibarguren, Izaskun; Villamarín, Antonio

2017-01-01

All the cells of higher organisms have the same DNA but not the same proteins. Each type of specialised cell that forms a tissue has its own pattern of gene expression and, consequently, it contains a particular set of proteins that determine its function. Here, we describe a laboratory exercise addressed to undergraduate students that aims to…

A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

ERIC Educational Resources Information Center

Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Schneider, Maria Paula Cruz; Ward, Richard John

2011-01-01

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from "Aequorea victoria" by a random mutagenesis strategy using error-prone polymerase…

Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.

PubMed

Swartz, Douglas J; Mok, Leo; Botta, Sri K; Singh, Anukriti; Altenberg, Guillermo A; Urbatsch, Ina L

2014-06-25

Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful.

Heteronuclear Multidimensional Protein NMR in a Teaching Laboratory

ERIC Educational Resources Information Center

Wright, Nathan T.

2016-01-01

Heteronuclear multidimensional NMR techniques are commonly used to study protein structure, function, and dynamics, yet they are rarely taught at the undergraduate level. Here, we describe a senior undergraduate laboratory where students collect, process, and analyze heteronuclear multidimensional NMR experiments using an unstudied Ig domain (Ig2…

Evolution of an ancient protein function involved in organized multicellularity in animals

PubMed Central

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-01

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which – the evolution of GKPID’s capacity to bind the cortical marker protein – can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals. DOI: http://dx.doi.org/10.7554/eLife.10147.001 PMID:26740169

The evolution and function of protein tandem repeats in plants.

PubMed

Schaper, Elke; Anisimova, Maria

2015-04-01

Sequence tandem repeats (TRs) are abundant in proteomes across all domains of life. For plants, little is known about their distribution or contribution to protein function. We exhaustively annotated TRs and studied the evolution of TR unit variations for all Ensembl plants. Using phylogenetic patterns of TR units, we detected conserved TRs with unit number and order preserved during evolution, and those TRs that have diverged via recent TR unit gains/losses. We correlated the mode of evolution of TRs to protein function. TR number was strongly correlated with proteome size, with about one-half of all TRs recognized as common protein domains. The majority of TRs have been highly conserved over long evolutionary distances, some since the separation of red algae and green plants c. 1.6 billion yr ago. Conversely, recurrent recent TR unit mutations were rare. Our results suggest that the first TRs by far predate the first plants, and that TR appearance is an ongoing process with similar rates across the plant kingdom. Interestingly, the few detected highly mutable TRs might provide a source of variation for rapid adaptation. In particular, such TRs are enriched in leucine-rich repeats (LRRs) commonly found in R genes, where TR unit gain/loss may facilitate resistance to emerging pathogens. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Biochemical Evolution of Iron and Copper Proteins, Substances Vital to Life

ERIC Educational Resources Information Center

Frieden, Earl

1974-01-01

Summarizes studies in the area of biochemical evolution of iron, copper, and heme proteins to provide an historical outline. Included are lists of major kinds of proteins and enzymes and charts illustrating electron flow in a cytochrome electron transport system and interconversion of jerrous to ferric ion in iron metabolism. (CC)

Rapid Evolution of Coral Proteins Responsible for Interaction with the Environment

PubMed Central

Matz, Mikhail V.; Bayer, Till; Aranda, Manuel; Buschiazzo, Emmanuel; DeSalvo, Michael K.; Lindquist, Erika; Szmant, Alina M.; Coffroth, Mary Alice; Medina, Mónica

2011-01-01

Background Corals worldwide are in decline due to climate change effects (e.g., rising seawater temperatures), pollution, and exploitation. The ability of corals to cope with these stressors in the long run depends on the evolvability of the underlying genetic networks and proteins, which remain largely unknown. A genome-wide scan for positively selected genes between related coral species can help to narrow down the search space considerably. Methodology/Principal Findings We screened a set of 2,604 putative orthologs from EST-based sequence datasets of the coral species Acropora millepora and Acropora palmata to determine the fraction and identity of proteins that may experience adaptive evolution. 7% of the orthologs show elevated rates of evolution. Taxonomically-restricted (i.e. lineage-specific) genes show a positive selection signature more frequently than genes that are found across many animal phyla. The class of proteins that displayed elevated evolutionary rates was significantly enriched for proteins involved in immunity and defense, reproduction, and sensory perception. We also found elevated rates of evolution in several other functional groups such as management of membrane vesicles, transmembrane transport of ions and organic molecules, cell adhesion, and oxidative stress response. Proteins in these processes might be related to the endosymbiotic relationship corals maintain with dinoflagellates in the genus Symbiodinium. Conclusion/Relevance This study provides a birds-eye view of the processes potentially underlying coral adaptation, which will serve as a foundation for future work to elucidate the rates, patterns, and mechanisms of corals' evolutionary response to global climate change. PMID:21633702

Rapid Evolution of Coral Proteins Responsible for Interaction with the Environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voolstra, Christian R.; Sunagawa, Shinichi; Matz, Mikhail V.

Background: Corals worldwide are in decline due to climate change effects (e.g., rising seawater temperatures), pollution, and exploitation. The ability of corals to cope with these stressors in the long run depends on the evolvability of the underlying genetic networks and proteins, which remain largely unknown. A genome-wide scan for positively selected genes between related coral species can help to narrow down the search space considerably. Methodology/Principal Findings: We screened a set of 2,604 putative orthologs from EST-based sequence datasets of the coral species Acropora millepora and Acropora palmata to determine the fraction and identity of proteins that may experiencemore » adaptive evolution. 7percent of the orthologs show elevated rates of evolution. Taxonomically-restricted (i.e. lineagespecific) genes show a positive selection signature more frequently than genes that are found across many animal phyla. The class of proteins that displayed elevated evolutionary rates was significantly enriched for proteins involved in immunity and defense, reproduction, and sensory perception. We also found elevated rates of evolution in several other functional groups such as management of membrane vesicles, transmembrane transport of ions and organic molecules, cell adhesion, and oxidative stress response. Proteins in these processes might be related to the endosymbiotic relationship corals maintain with dinoflagellates in the genus Symbiodinium. Conclusion/Relevance: This study provides a birds-eye view of the processes potentially underlying coral adaptation, which will serve as a foundation for future work to elucidate the rates, patterns, and mechanisms of corals? evolutionary response to global climate change.« less

The origin of polynucleotide-directed protein synthesis

NASA Technical Reports Server (NTRS)

Orgel, Leslie E.

1989-01-01

If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that at first the simple attachment of amino acids to the 2'(3') termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

Evolution and characterization of a new reversibly photoswitching chromogenic protein, Dathail

DOE PAGES

Langan, Patricia S.; Close, Devin W.; Coates, Leighton; ...

2016-03-18

In this paper, we report the engineering of a new reversibly switching chromogenic protein, Dathail. Dathail was evolved from the extremely thermostable fluorescent proteins thermal green protein (TGP) and eCGP123 using directed evolution and ratiometric sorting. Dathail has two spectrally distinct chromogenic states with low quantum yields, corresponding to absorbance in a ground state with a maximum at 389 nm, and a photo-induced metastable state with a maximum at 497 nm. In contrast to all previously described photoswitchable proteins, both spectral states of Dathail are non-fluorescent. The photo-induced chromogenic state of Dathail has a lifetime of ~ 50 min atmore » 293 K and pH 7.5 as measured by UV–Vis spectrophotometry, returning to the ground state through thermal relaxation. X-ray crystallography provided structural insights supporting a change in conformation and coordination in the chromophore pocket as being responsible for Dathail's photoswitching. Neutron crystallography, carried out for the first time on a protein from the green fluorescent protein family, showed a distribution of hydrogen atoms revealing protonation of the chromophore 4-hydroxybenzyl group in the ground state. Additionally, the neutron structure also supports the hypothesis that the photo-induced proton transfer from the chromophore occurs through water-mediated proton relay into the bulk solvent. Beyond its spectroscopic curiosity, Dathail has several characteristics that are improvements for applications, including low background fluorescence, large spectral separation, rapid switching time, and the ability to switch many times. Therefore, Dathail is likely to be extremely useful in the quickly developing fields of imaging and biosensors, including photochromic Förster resonance energy transfer, high-resolution microscopy, and live tracking within the cell.« less

Simulation of the 3-D Evolution of Electron Scale Magnetic Reconnection - Motivated by Laboratory Experiments Predictions for MMS

NASA Astrophysics Data System (ADS)

Buechner, J.; Jain, N.; Sharma, A.

2013-12-01

The four s/c of the Magnetospheric Multiscale (MMS) mission, to be launched in 2014, will use the Earth's magnetosphere as a laboratory to study the microphysics of three fundamental plasma processes. One of them is magnetic reconnection, an essentially multi-scale process. While laboratory experiments and past theoretical investigations have shown that important processes necessary to understand magnetic reconnection take place at electron scales the MMS mission for the first time will be able to resolve these scales by in space observations. For the measurement strategy of MMS it is important to make specific predictions of the behavior of current sheets with a thickness of the order of the electron skin depth which play an important role in the evolution of collisionless magnetic reconnection. Since these processes are highly nonlinear and non-local numerical simulation is needed to specify the current sheet evolution. Here we present new results about the nonlinear evolution of electron-scale current sheets starting from the linear stage and using 3-D electron-magnetohydrodynamic (EMHD) simulations. The growth rates of the simulated instabilities compared well with the growth rates obtained from linear theory. Mechanisms and conditions of the formation of flux ropes and of current filamentation will be discussed in comparison with the results of fully kinetic simulations. In 3D the X- and O-point configurations of the magnetic field formed in reconnection planes alternate along the out-of-reconnection-plane direction with the wavelength of the unstable mode. In the presence of multiple reconnection sites, the out-of-plane magnetic field can develop nested structure of quadrupoles in reconnection planes, similar to the 2-D case, but now with variations in the out-of-plane direction. The structures of the electron flow and magnetic field in 3-D simulations will be compared with those in 2-D simulations to discriminate the essentially 3D features. We also discuss

A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA.

PubMed

Aydin, Suleyman

2015-10-01

Playing a critical role in the metabolic homeostasis of living systems, the circulating concentrations of peptides/proteins are influenced by a variety of patho-physiological events. These peptide/protein concentrations in biological fluids are measured using various methods, the most common of which is enzymatic immunoassay EIA/ELISA and which guide the clinicians in diagnosing and monitoring diseases that inflict biological systems. All the techniques where enzymes are employed to show antigen-antibody reactions are generally referred to as enzymatic immunoassay EIA/ELISA method. Since the basic principles of EIA and ELISA are the same. The main objective of this review is to present an overview of the historical journey that had led to the invention of EIA/ELISA, an indispensible method for medical and research laboratories, types of ELISA developed after its invention [direct (the first ELISA method invented), indirect, sandwich and competitive methods], problems encountered during peptide/protein analyses (pre-analytical, analytical and post-analytical), rules to be followed to prevent these problems, and our laboratory experience of more than 15 years. Copyright © 2015 Elsevier Inc. All rights reserved.

Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

NASA Astrophysics Data System (ADS)

He, Yi-Ming; Ma, Bin-Guang

2016-05-01

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

Evolution of intrinsic disorder in eukaryotic proteins.

PubMed

Ahrens, Joseph B; Nunez-Castilla, Janelle; Siltberg-Liberles, Jessica

2017-09-01

Conformational flexibility conferred though regions of intrinsic structural disorder allows proteins to behave as dynamic molecules. While it is well-known that intrinsically disordered regions can undergo disorder-to-order transitions in real-time as part of their function, we also are beginning to learn more about the dynamics of disorder-to-order transitions along evolutionary time-scales. Intrinsically disordered regions endow proteins with functional promiscuity, which is further enhanced by the ability of some of these regions to undergo real-time disorder-to-order transitions. Disorder content affects gene retention after whole genome duplication, but it is not necessarily conserved. Altered patterns of disorder resulting from evolutionary disorder-to-order transitions indicate that disorder evolves to modify function through refining stability, regulation, and interactions. Here, we review the evolution of intrinsically disordered regions in eukaryotic proteins. We discuss the interplay between secondary structure and disorder on evolutionary time-scales, the importance of disorder for eukaryotic proteome expansion and functional divergence, and the evolutionary dynamics of disorder.

The evolution of phenotypic integration: How directional selection reshapes covariation in mice.

PubMed

Penna, Anna; Melo, Diogo; Bernardi, Sandra; Oyarzabal, Maria Inés; Marroig, Gabriel

2017-10-01

Variation is the basis for evolution, and understanding how variation can evolve is a central question in biology. In complex phenotypes, covariation plays an even more important role, as genetic associations between traits can bias and alter evolutionary change. Covariation can be shaped by complex interactions between loci, and this genetic architecture can also change during evolution. In this article, we analyzed mouse lines experimentally selected for changes in size to address the question of how multivariate covariation changes under directional selection, as well as to identify the consequences of these changes to evolution. Selected lines showed a clear restructuring of covariation in their cranium and, instead of depleting their size variation, these lines increased their magnitude of integration and the proportion of variation associated with the direction of selection. This result is compatible with recent theoretical works on the evolution of covariation that take the complexities of genetic architecture into account. This result also contradicts the traditional view of the effects of selection on available covariation and suggests a much more complex view of how populations respond to selection. © 2017 The Author(s). Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.

Functional mapping of protein-protein interactions in an enzyme complex by directed evolution.

PubMed

Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

2014-01-01

The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84-90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84-86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.

Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

PubMed Central

Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

2014-01-01

The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes. PMID:25551646

Spectral evolution of weakly nonlinear random waves: kinetic description vs direct numerical simulations

NASA Astrophysics Data System (ADS)

Annenkov, Sergei; Shrira, Victor

2016-04-01

We study numerically the long-term evolution of water wave spectra without wind forcing, using three different models, aiming at understanding the role of different sets of assumptions. The first model is the classical Hasselmann kinetic equation (KE). We employ the WRT code kindly provided by G. van Vledder. Two other models are new. As the second model, we use the generalised kinetic equation (gKE), derived without the assumption of quasi-stationarity. Thus, unlike the KE, the gKE is valid in the cases when a wave spectrum is changing rapidly (e.g. at the initial stage of evolution of a narrow spectrum). However, the gKE employs the same statistical closure as the KE. The third model is based on the Zakharov integrodifferential equation for water waves and does not depend on any statistical assumptions. Since the Zakharov equation plays the role of the primitive equation of the theory of wave turbulence, we refer to this model as direct numerical simulation of spectral evolution (DNS-ZE). For initial conditions, we choose two narrow-banded spectra with the same frequency distribution (a JONSWAP spectrum with high peakedness γ = 6) and different degrees of directionality. These spectra are from the set of observations collected in a directional wave tank by Onorato et al (2009). Spectrum A is very narrow in angle (corresponding to N = 840 in the cosN directional model). Spectrum B is initially wider in angle (corresponds to N = 24). Short-term evolution of both spectra (O(102) wave periods) has been studied numerically by Xiao et al (2013) using two other approaches (broad-band modified nonlinear Schrödinger equation and direct numerical simulation based on the high-order spectral method). We use these results to verify the initial stage of our DNS-ZE simulations. However, the advantage of the DNS-ZE method is that it allows to study long-term spectral evolution (up to O(104) periods), which was previously possible only with the KE. In the short-term evolution

Rapid and Adaptable Measurement of Protein Thermal Stability by Differential Scanning Fluorimetry: Updating a Common Biochemical Laboratory Experiment

ERIC Educational Resources Information Center

Johnson, R. Jeremy; Savas, Christopher J.; Kartje, Zachary; Hoops, Geoffrey C.

2014-01-01

Measurement of protein denaturation and protein folding is a common laboratory technique used in undergraduate biochemistry laboratories. Differential scanning fluorimetry (DSF) provides a rapid, sensitive, and general method for measuring protein thermal stability in an undergraduate biochemistry laboratory. In this method, the thermal…

Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

PubMed Central

He, Yi-Ming; Ma, Bin-Guang

2016-01-01

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

Conflict RNA modification, host-parasite co-evolution, and the origins of DNA and DNA-binding proteins1.

PubMed

McLaughlin, Paul J; Keegan, Liam P

2014-08-01

Nearly 150 different enzymatically modified forms of the four canonical residues in RNA have been identified. For instance, enzymes of the ADAR (adenosine deaminase acting on RNA) family convert adenosine residues into inosine in cellular dsRNAs. Recent findings show that DNA endonuclease V enzymes have undergone an evolutionary transition from cleaving 3' to deoxyinosine in DNA and ssDNA to cleaving 3' to inosine in dsRNA and ssRNA in humans. Recent work on dsRNA-binding domains of ADARs and other proteins also shows that a degree of sequence specificity is achieved by direct readout in the minor groove. However, the level of sequence specificity observed is much less than that of DNA major groove-binding helix-turn-helix proteins. We suggest that the evolution of DNA-binding proteins following the RNA to DNA genome transition represents the major advantage that DNA genomes have over RNA genomes. We propose that a hypothetical RNA modification, a RRAR (ribose reductase acting on genomic dsRNA) produced the first stretches of DNA in RNA genomes. We discuss why this is the most satisfactory explanation for the origin of DNA. The evolution of this RNA modification and later steps to DNA genomes are likely to have been driven by cellular genome co-evolution with viruses and intragenomic parasites. RNA modifications continue to be involved in host-virus conflicts; in vertebrates, edited cellular dsRNAs with inosine-uracil base pairs appear to be recognized as self RNA and to suppress activation of innate immune sensors that detect viral dsRNA.

Inquiry, Investigation, and Communication in the Student-Directed Laboratory.

ERIC Educational Resources Information Center

Janners, Martha Y.

1988-01-01

Describes how to organize a student-directed laboratory investigation which is based on amphibian metamorphosis, lasts for nearly a term, and involves extensive group effort. Explains the assignment, student response and opinion, formal paper, and instructor responsibilities. (RT)

Understanding the evolution of luminescent gold quantum clusters in protein templates.

PubMed

Chaudhari, Kamalesh; Xavier, Paulrajpillai Lourdu; Pradeep, Thalappil

2011-11-22

We show that the time-dependent biomineralization of Au(3+) by native lactoferrin (NLf) and bovine serum albumin (BSA) resulting in near-infrared (NIR) luminescent gold quantum clusters (QCs) occurs through a protein-bound Au(1+) intermediate and subsequent emergence of free protein. The evolution was probed by diverse tools, principally, using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), X-ray photoelectron spectroscopy (XPS), and photoluminescence spectroscopy (PL). The importance of alkaline pH in the formation of clusters was probed. At neutral pH, a Au(1+)-protein complex was formed (starting from Au(3+)) with the binding of 13-14 gold atoms per protein. When the pH was increased above 12, these bound gold ions were further reduced to Au(0) and nucleation and growth of cluster commenced, which was corroborated by the beginning of emission; at this point, the number of gold atoms per protein was ~25, suggesting the formation of Au(25). During the cluster evolution, at certain time intervals, for specific molar ratios of gold and protein, occurrence of free protein was noticed in the mass spectra, suggesting a mixture of products and gold ion redistribution. By providing gold ions at specific time of the reaction, monodispersed clusters with enhanced luminescence could be obtained, and the available quantity of free protein could be utilized efficiently. Monodispersed clusters would be useful in obtaining single crystals of protein-protected noble metal quantum clusters where homogeneity of the system is of primary concern. © 2011 American Chemical Society

Essentiality Is a Strong Determinant of Protein Rates of Evolution during Mutation Accumulation Experiments in Escherichia coli.

PubMed

Alvarez-Ponce, David; Sabater-Muñoz, Beatriz; Toft, Christina; Ruiz-González, Mario X; Fares, Mario A

2016-09-26

The Neutral Theory of Molecular Evolution is considered the most powerful theory to understand the evolutionary behavior of proteins. One of the main predictions of this theory is that essential proteins should evolve slower than dispensable ones owing to increased selective constraints. Comparison of genomes of different species, however, has revealed only small differences between the rates of evolution of essential and nonessential proteins. In some analyses, these differences vanish once confounding factors are controlled for, whereas in other cases essentiality seems to have an independent, albeit small, effect. It has been argued that comparing relatively distant genomes may entail a number of limitations. For instance, many of the genes that are dispensable in controlled lab conditions may be essential in some of the conditions faced in nature. Moreover, essentiality can change during evolution, and rates of protein evolution are simultaneously shaped by a variety of factors, whose individual effects are difficult to isolate. Here, we conducted two parallel mutation accumulation experiments in Escherichia coli, during 5,500-5,750 generations, and compared the genomes at different points of the experiments. Our approach (a short-term experiment, under highly controlled conditions) enabled us to overcome many of the limitations of previous studies. We observed that essential proteins evolved substantially slower than nonessential ones during our experiments. Strikingly, rates of protein evolution were only moderately affected by expression level and protein length. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Models of Protocellular Structure, Function and Evolution

NASA Technical Reports Server (NTRS)

New, Michael H.; Pohorille, Andrew; Szostak, Jack W.; Keefe, Tony; Lanyi, Janos K.; DeVincenzi, Donald L. (Technical Monitor)

2001-01-01

In the absence of any record of protocells, the most direct way to test our understanding, of the origin of cellular life is to construct laboratory models that capture important features of protocellular systems. Such efforts are currently underway in a collaborative project between NASA-Ames, Harvard Medical School and University of California. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures. The centerpiece of this project is a method for the in vitro evolution of protein enzymes toward arbitrary catalytic targets. A similar approach has already been developed for nucleic acids in which a small number of functional molecules are selected from a large, random population of candidates. The selected molecules are next vastly multiplied using the polymerase chain reaction.

Modeling the evolution of protein domain architectures using maximum parsimony.

PubMed

Fong, Jessica H; Geer, Lewis Y; Panchenko, Anna R; Bryant, Stephen H

2007-02-09

Domains are basic evolutionary units of proteins and most proteins have more than one domain. Advances in domain modeling and collection are making it possible to annotate a large fraction of known protein sequences by a linear ordering of their domains, yielding their architecture. Protein domain architectures link evolutionarily related proteins and underscore their shared functions. Here, we attempt to better understand this association by identifying the evolutionary pathways by which extant architectures may have evolved. We propose a model of evolution in which architectures arise through rearrangements of inferred precursor architectures and acquisition of new domains. These pathways are ranked using a parsimony principle, whereby scenarios requiring the fewest number of independent recombination events, namely fission and fusion operations, are assumed to be more likely. Using a data set of domain architectures present in 159 proteomes that represent all three major branches of the tree of life allows us to estimate the history of over 85% of all architectures in the sequence database. We find that the distribution of rearrangement classes is robust with respect to alternative parsimony rules for inferring the presence of precursor architectures in ancestral species. Analyzing the most parsimonious pathways, we find 87% of architectures to gain complexity over time through simple changes, among which fusion events account for 5.6 times as many architectures as fission. Our results may be used to compute domain architecture similarities, for example, based on the number of historical recombination events separating them. Domain architecture "neighbors" identified in this way may lead to new insights about the evolution of protein function.

Laboratory Directed Research and Development FY2001 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Ayat, R

2002-06-20

Established by Congress in 1991, the Laboratory Directed Research and Development (LDRD) Program provides the Department of Energy (DOE)/National Nuclear Security Administration (NNSA) laboratories, like Lawrence Livermore National Laboratory (LLNL or the Laboratory), with the flexibility to invest up to 6% of their budget in long-term, high-risk, and potentially high payoff research and development (R&D) activities to support the DOE/NNSA's national security missions. By funding innovative R&D, the LDRD Program at LLNL develops and extends the Laboratory's intellectual foundations and maintains its vitality as a premier research institution. As proof of the Program's success, many of the research thrusts thatmore » started many years ago under LDRD sponsorship are at the core of today's programs. The LDRD Program, which serves as a proving ground for innovative ideas, is the Laboratory's most important single resource for fostering excellent science and technology for today's needs and tomorrow's challenges. Basic and applied research activities funded by LDRD enhance the Laboratory's core strengths, driving its technical vitality to create new capabilities that enable LLNL to meet DOE/NNSA's national security missions. The Program also plays a key role in building a world-class multidisciplinary workforce by engaging the Laboratory's best researchers, recruiting its future scientists and engineers, and promoting collaborations with all sectors of the larger scientific community.« less

Directed evolution of a model primordial enzyme provides insights into the development of the genetic code.

PubMed

Müller, Manuel M; Allison, Jane R; Hongdilokkul, Narupat; Gaillon, Laurent; Kast, Peter; van Gunsteren, Wilfred F; Marlière, Philippe; Hilvert, Donald

2013-01-01

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.

Development of a Green Fluorescent Protein-Based Laboratory Curriculum

ERIC Educational Resources Information Center

Larkin, Patrick D.; Hartberg, Yasha

2005-01-01

A laboratory curriculum has been designed for an undergraduate biochemistry course that focuses on the investigation of the green fluorescent protein (GFP). The sequence of procedures extends from analysis of the DNA sequence through PCR amplification, recombinant plasmid DNA synthesis, bacterial transformation, expression, isolation, and…

Studying Sexual Aggression: A Review of the Evolution and Validity of Laboratory Paradigms

PubMed Central

Davis, Kelly Cue; George, William H.; Nagayama Hall, Gordon C.; Parrott, Dominic J.; Tharp, Andra Teten; Stappenbeck, Cynthia A.

2018-01-01

Objective Researchers have endeavored for decades to develop and implement experimental assessments of sexual aggression and its precursors to capitalize on the many scientific advantages offered by laboratory experiments, such as rigorous control of key variables and identification of causal relationships. The purpose of this review is to provide an overview of and commentary on the evolution of these laboratory-based methods. Conclusions To date, two primary types of sexual aggression laboratory studies have been developed: those that involve behavioral analogues of sexual aggression and those that assess postulated precursors to sexually aggressive behavior. Although the study of sexual aggression in the laboratory is fraught with methodological challenges, validity concerns, and ethical considerations, advances in the field have resulted in greater methodological rigor, more precise dependent measures, and improved experimental validity, reliability, and realism. Because highly effective sexual aggression prevention strategies remain elusive, continued laboratory-based investigation of sexual aggression coupled with translation of critical findings to the development and modification of sexual aggression prevention programs remains an important task for the field. PMID:29675289

ProteomeVis: a web app for exploration of protein properties from structure to sequence evolution across organisms' proteomes.

PubMed

Razban, Rostam M; Gilson, Amy I; Durfee, Niamh; Strobelt, Hendrik; Dinkla, Kasper; Choi, Jeong-Mo; Pfister, Hanspeter; Shakhnovich, Eugene I

2018-05-08

Protein evolution spans time scales and its effects span the length of an organism. A web app named ProteomeVis is developed to provide a comprehensive view of protein evolution in the S. cerevisiae and E. coli proteomes. ProteomeVis interactively creates protein chain graphs, where edges between nodes represent structure and sequence similarities within user-defined ranges, to study the long time scale effects of protein structure evolution. The short time scale effects of protein sequence evolution are studied by sequence evolutionary rate (ER) correlation analyses with protein properties that span from the molecular to the organismal level. We demonstrate the utility and versatility of ProteomeVis by investigating the distribution of edges per node in organismal protein chain universe graphs (oPCUGs) and putative ER determinants. S. cerevisiae and E. coli oPCUGs are scale-free with scaling constants of 1.79 and 1.56, respectively. Both scaling constants can be explained by a previously reported theoretical model describing protein structure evolution (Dokholyan et al., 2002). Protein abundance most strongly correlates with ER among properties in ProteomeVis, with Spearman correlations of -0.49 (p-value<10-10) and -0.46 (p-value<10-10) for S. cerevisiae and E. coli, respectively. This result is consistent with previous reports that found protein expression to be the most important ER determinant (Zhang and Yang, 2015). ProteomeVis is freely accessible at http://proteomevis.chem.harvard.edu. Supplementary data are available at Bioinformatics. shakhnovich@chemistry.harvard.edu.

Massively Convergent Evolution for Ribosomal Protein Gene Content in Plastid and Mitochondrial Genomes

PubMed Central

Maier, Uwe-G; Zauner, Stefan; Woehle, Christian; Bolte, Kathrin; Hempel, Franziska; Allen, John F.; Martin, William F.

2013-01-01

Plastid and mitochondrial genomes have undergone parallel evolution to encode the same functional set of genes. These encode conserved protein components of the electron transport chain in their respective bioenergetic membranes and genes for the ribosomes that express them. This highly convergent aspect of organelle genome evolution is partly explained by the redox regulation hypothesis, which predicts a separate plastid or mitochondrial location for genes encoding bioenergetic membrane proteins of either photosynthesis or respiration. Here we show that convergence in organelle genome evolution is far stronger than previously recognized, because the same set of genes for ribosomal proteins is independently retained by both plastid and mitochondrial genomes. A hitherto unrecognized selective pressure retains genes for the same ribosomal proteins in both organelles. On the Escherichia coli ribosome assembly map, the retained proteins are implicated in 30S and 50S ribosomal subunit assembly and initial rRNA binding. We suggest that ribosomal assembly imposes functional constraints that govern the retention of ribosomal protein coding genes in organelles. These constraints are subordinate to redox regulation for electron transport chain components, which anchor the ribosome to the organelle genome in the first place. As organelle genomes undergo reduction, the rRNAs also become smaller. Below size thresholds of approximately 1,300 nucleotides (16S rRNA) and 2,100 nucleotides (26S rRNA), all ribosomal protein coding genes are lost from organelles, while electron transport chain components remain organelle encoded as long as the organelles use redox chemistry to generate a proton motive force. PMID:24259312

Laboratory Directed Research and Development Annual Report FY 2017

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sullivan, Kelly O.

A national laboratory must establish and maintain an environment in which creativity and innovation are encouraged and supported in order to fulfill its missions and remain viable in the long term. As such, multiprogram laboratories are given discretion to allocate a percentage of their operating budgets to support research and development projects that align to PNNL’s and DOE’s missions and support the missions of other federal agencies, including DHS, DOD, and others. DOE Order 413.2C sets forth DOE’s Laboratory Directed Research and Development (LDRD) policy and guidelines for DOE multiprogram laboratories, and it authorizes the national laboratories to allocate upmore » to 6 percent of their operating budgets to fund the program. LDRD is innovative research and development, selected by the Laboratory Director or his/her designee, for the purpose of maintaining the scientific and technological vitality of the Laboratory. The projects supported by LDRD funding all have demonstrable ties to DOE/DHS missions and may also be relevant to the missions of other federal agencies that sponsor work at the Laboratory. The program plays a key role in attracting the best and brightest scientific staff, which is needed to serve the highest priority DOE mission objectives. Individual project reports comprise the bulk of this LDRD report. The Laboratory focuses its LDRD research on scientific assets that often address more than one scientific discipline.« less

Laboratory Directed Research and Development Annual Report FY 2016

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sullivan, Kelly O.

A national laboratory must establish and maintain an environment in which creativity and innovation are encouraged and supported in order to fulfill its missions and remain viable in the long term. As such, multiprogram laboratories are given discretion to allocate a percentage of their operating budgets to support research and development projects that align to PNNL’s and DOE’s missions and support the missions of other federal agencies, including DHS, DOD, and others. DOE Order 413.2C sets forth DOE’s Laboratory Directed Research and Development (LDRD) policy and guidelines for DOE multiprogram laboratories, and it authorizes the national laboratories to allocate upmore » to 6 percent of their operating budgets to fund the program. LDRD is innovative research and development, selected by the Laboratory Director or his/her designee, for the purpose of maintaining the scientific and technological vitality of the Laboratory. The projects supported by LDRD funding all have demonstrable ties to DOE/DHS missions and may also be relevant to the missions of other federal agencies that sponsor work at the Laboratory. The program plays a key role in attracting the best and brightest scientific staff, which is needed to serve the highest priority DOE mission objectives. Individual project reports comprise the bulk of this LDRD report. The Laboratory focuses its LDRD research on scientific assets that often address more than one scientific discipline.« less

Evolution of group-wise cooperation: Is direct reciprocity insufficient?

PubMed

Kurokawa, Shun; Ihara, Yasuo

2017-02-21

Group-wise cooperation, or cooperation among three or more individuals, is an integral part of human societies. It is likely that group-wise cooperation also played a crucial role in the survival of early hominins, who were confronted with novel environmental challenges, long before the emergence of Homo sapiens. However, previous theoretical and empirical studies, focusing mainly on modern humans, have tended to suggest that evolution of cooperation in sizable groups cannot be explained by simple direct reciprocity and requires some additional mechanisms (reputation, punishment, etc.), which are cognitively too demanding for early hominins. As a partial resolution of the paradox, our recent analysis of a stochastic evolutionary model, which considers the effect of random drift, has revealed that evolution of group-wise cooperation is more likely to occur in larger groups when an individual's share of the benefit produced by one cooperator does not decrease with increasing group size (i.e., goods are non-rivalrous). In this paper, we further extend our previous analysis to explore possible consequences of introducing rare mistakes in behavior or imperfect information about behavior of others on the model outcome. Analyses of the extended models show that evolution of group-wise cooperation can be facilitated by large group size even when individuals intending to cooperate sometimes fail to do so or when all the information about the past behavior of group members is not available. We argue, therefore, that evolution of cooperation in sizable groups does not necessarily require other mechanisms than direct reciprocity if the goods to be produced via group-wise cooperation are non-rivalrous. Copyright © 2016 Elsevier Ltd. All rights reserved.

Laboratory Directed Research and Development FY 1998 Progress Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

John Vigil; Kyle Wheeler

This is the FY 1998 Progress Report for the Laboratory Directed Research and Development (LDRD) Program at Los Alamos National Laboratory. It gives an overview of the LDRD Program, summarizes work done on individual research projects, relates the projects to major Laboratory program sponsors, and provides an index to the principle investigators. Project summaries are grouped by their LDRD component: Competency Development, Program Development, and Individual Projects. Within each component, they are further grouped into nine technical categories: (1) materials science, (2) chemistry, (3) mathematics and computational science, (4) atomic, molecular, optical, and plasma physics, fluids, and particle beams, (5)more » engineering science, (6) instrumentation and diagnostics, (7) geoscience, space science, and astrophysics, (8) nuclear and particle physics, and (9) bioscience.« less

Laboratory directed research and development: FY 1997 progress report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vigil, J.; Prono, J.

1998-05-01

This is the FY 1997 Progress Report for the Laboratory Directed Research and Development (LDRD) program at Los Alamos National Laboratory. It gives an overview of the LDRD program, summarizes work done on individual research projects, relates the projects to major Laboratory program sponsors, and provides an index to the principal investigators. Project summaries are grouped by their LDRD component: Competency Development, Program Development, and Individual Projects. Within each component, they are further grouped into nine technical categories: (1) materials science, (2) chemistry, (3) mathematics and computational science, (4) atomic and molecular physics and plasmas, fluids, and particle beams, (5)more » engineering science, (6) instrumentation and diagnostics, (7) geoscience, space science, and astrophysics, (8) nuclear and particle physics, and (9) bioscience.« less

Evol and ProDy for bridging protein sequence evolution and structural dynamics

PubMed Central

Mao, Wenzhi; Liu, Ying; Chennubhotla, Chakra; Lezon, Timothy R.; Bahar, Ivet

2014-01-01

Correlations between sequence evolution and structural dynamics are of utmost importance in understanding the molecular mechanisms of function and their evolution. We have integrated Evol, a new package for fast and efficient comparative analysis of evolutionary patterns and conformational dynamics, into ProDy, a computational toolbox designed for inferring protein dynamics from experimental and theoretical data. Using information-theoretic approaches, Evol coanalyzes conservation and coevolution profiles extracted from multiple sequence alignments of protein families with their inferred dynamics. Availability and implementation: ProDy and Evol are open-source and freely available under MIT License from http://prody.csb.pitt.edu/. Contact: bahar@pitt.edu PMID:24849577

Textural Evolution During Micro Direct Metal Deposition of NiTi Alloy

NASA Astrophysics Data System (ADS)

Khademzadeh, Saeed; Bariani, Paolo F.; Bruschi, Stefania

2018-03-01

In this research, a micro direct metal deposition process, newly developed as a potential method for micro additive manufacturing was used to fabricate NiTi builds. The effect of scanning strategy on grain growth and textural evolution was investigated using scanning electron microscope equipped with electron backscattered diffraction detector. Investigations showed that, the angle between the successive single tracks has an important role in grain size distribution and textural evolution of NiTi phase. Unidirectional laser beam scanning pattern developed a fiber texture; conversely, a backward and forward scanning pattern developed a strong < {100} > ‖‖ RD texture on the surface of NiTi cubic samples produced by micro direct metal deposition.

Textural Evolution During Micro Direct Metal Deposition of NiTi Alloy

NASA Astrophysics Data System (ADS)

Khademzadeh, Saeed; Bariani, Paolo F.; Bruschi, Stefania

2018-07-01

In this research, a micro direct metal deposition process, newly developed as a potential method for micro additive manufacturing was used to fabricate NiTi builds. The effect of scanning strategy on grain growth and textural evolution was investigated using scanning electron microscope equipped with electron backscattered diffraction detector. Investigations showed that, the angle between the successive single tracks has an important role in grain size distribution and textural evolution of NiTi phase. Unidirectional laser beam scanning pattern developed a fiber texture; conversely, a backward and forward scanning pattern developed a strong < {100} > ‖‖ RD texture on the surface of NiTi cubic samples produced by micro direct metal deposition.

Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years

PubMed Central

Nicosia, Aldo; Maggio, Teresa; Costa, Salvatore; Salamone, Monica; Tagliavia, Marcello; Mazzola, Salvatore; Gianguzza, Fabrizio; Cuttitta, Angela

2016-01-01

Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations. PMID:26957029

Evolution of organic molecules under Mars-like UV radiation conditions in space and laboratory

NASA Astrophysics Data System (ADS)

Rouquette, L.; Stalport, F.; Cottin, H.; Coll, P.; Szopa, C.; Saiagh, K.; Poch, O.; Khalaf, D.; Chaput, D.; Grira, K.; Dequaire, T.

2017-09-01

The detection and identification of organic molecules at Mars are of prime importance, as some of these molecules are life precursors and components. While in situ planetary missions are searching for them, it is essential to understand how organic molecules evolve and are preserved at the surface of Mars. Indeed the harsh conditions of the environment of Mars such as ultraviolet (UV) radiation or oxidative processes could explain the low abundance and diversity of organic molecules detected by now [1]. In order to get a better understanding of the evolution of organic matter at the surface of Mars, we exposed organic molecules under a Mars-like UV radiation environment. Similar organic samples were exposed to the Sun radiation, outside the International Space Station (ISS), and under a UV lamp (martian pressure and temperature conditions) in the laboratory. In both experiments, organic molecules tend to photodegrade under Mars-like UV radiation. Minerals, depending on their nature, can protect or accelerate the degradation of organic molecules. For some molecules, new products, possibly photoresistant, seem to be produced. Finally, experimenting in space allow us to get close to in situ conditions and to validate our laboratory experiment while the laboratory experiment is essential to study the evolution of a large amount and diversity of organic molecules.

A Laboratory Exercise to Illustrate Protein-Membrane Interactions

ERIC Educational Resources Information Center

Weers, Paul M. M.; Prenner, Elmar J.; Curic, Spomenka; Lohmeier-Vogel, Elke M.

2016-01-01

The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6-tagged human apolipoprotein A-I (apoA-I) with Ni-NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre-dried phospholipid films. When apoA-I is added to the MLV,…

Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.

PubMed

Renata, Hans; Wang, Z Jane; Arnold, Frances H

2015-03-09

High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature's known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Essentiality Is a Strong Determinant of Protein Rates of Evolution during Mutation Accumulation Experiments in Escherichia coli

PubMed Central

Alvarez-Ponce, David; Sabater-Muñoz, Beatriz; Toft, Christina; Ruiz-González, Mario X.; Fares, Mario A.

2016-01-01

Abstract The Neutral Theory of Molecular Evolution is considered the most powerful theory to understand the evolutionary behavior of proteins. One of the main predictions of this theory is that essential proteins should evolve slower than dispensable ones owing to increased selective constraints. Comparison of genomes of different species, however, has revealed only small differences between the rates of evolution of essential and nonessential proteins. In some analyses, these differences vanish once confounding factors are controlled for, whereas in other cases essentiality seems to have an independent, albeit small, effect. It has been argued that comparing relatively distant genomes may entail a number of limitations. For instance, many of the genes that are dispensable in controlled lab conditions may be essential in some of the conditions faced in nature. Moreover, essentiality can change during evolution, and rates of protein evolution are simultaneously shaped by a variety of factors, whose individual effects are difficult to isolate. Here, we conducted two parallel mutation accumulation experiments in Escherichia coli, during 5,500–5,750 generations, and compared the genomes at different points of the experiments. Our approach (a short-term experiment, under highly controlled conditions) enabled us to overcome many of the limitations of previous studies. We observed that essential proteins evolved substantially slower than nonessential ones during our experiments. Strikingly, rates of protein evolution were only moderately affected by expression level and protein length. PMID:27566759

The evolution of phenotypic integration: How directional selection reshapes covariation in mice

PubMed Central

Penna, Anna; Melo, Diogo; Bernardi, Sandra; Oyarzabal, Maria Inés; Marroig, Gabriel

2017-01-01

Abstract Variation is the basis for evolution, and understanding how variation can evolve is a central question in biology. In complex phenotypes, covariation plays an even more important role, as genetic associations between traits can bias and alter evolutionary change. Covariation can be shaped by complex interactions between loci, and this genetic architecture can also change during evolution. In this article, we analyzed mouse lines experimentally selected for changes in size to address the question of how multivariate covariation changes under directional selection, as well as to identify the consequences of these changes to evolution. Selected lines showed a clear restructuring of covariation in their cranium and, instead of depleting their size variation, these lines increased their magnitude of integration and the proportion of variation associated with the direction of selection. This result is compatible with recent theoretical works on the evolution of covariation that take the complexities of genetic architecture into account. This result also contradicts the traditional view of the effects of selection on available covariation and suggests a much more complex view of how populations respond to selection. PMID:28685813

Regolith evolution in the laboratory - Scaling dissimilar comminution experiments

NASA Technical Reports Server (NTRS)

Cintala, Mark J.; Horz, Friedrich

1990-01-01

Repeated impacts into fragmental targets simulating unconsolidated debris on planetary surfaces have provided empirical insight into the evolution of planetary regoliths. The techniques of dimensional analysis have been employed to quantify and examine the relationships between the more important variables in the evolution of these experimental regoliths. Application of this method to the results of 10 experimental series shows that the quantity of comminuted target mass is directly proportional to (1) the number of impacts, (2) the diameter of the projectile, (3) the mean size of the crystals, (4) the mean grain size of the evolving regolith, (5) the total target mass, (6) the impactor density, and (7) the ratio of the impact velocity to the velocity of sound in the target rock. The comminuted mass is inversely proportional to the density of the target rock and the sorting of the regolith.

Protein Flexibility Facilitates Quaternary Structure Assembly and Evolution

PubMed Central

Marsh, Joseph A.; Teichmann, Sarah A.

2014-01-01

The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also commonly assemble into complexes with diverse quaternary structure arrangements. Here we investigate how the flexibility of individual protein chains influences the assembly and evolution of protein complexes. We find that flexibility appears to be particularly conducive to the formation of heterologous (i.e., asymmetric) intersubunit interfaces. This leads to a strong association between subunit flexibility and homomeric complexes with cyclic and asymmetric quaternary structure topologies. Similarly, we also observe that the more nonhomologous subunits that assemble together within a complex, the more flexible those subunits tend to be. Importantly, these findings suggest that subunit flexibility should be closely related to the evolutionary history of a complex. We confirm this by showing that evolutionarily more recent subunits are generally more flexible than evolutionarily older subunits. Finally, we investigate the very different explorations of quaternary structure space that have occurred in different evolutionary lineages. In particular, the increased flexibility of eukaryotic proteins appears to enable the assembly of heteromeric complexes with more unique components. PMID:24866000

The Evolution of MicroRNA Pathway Protein Components in Cnidaria

PubMed Central

Moran, Yehu; Praher, Daniela; Fredman, David; Technau, Ulrich

2013-01-01

In the last decade, it became evident that posttranscriptional regulation of gene expression by microRNAs is a central biological process in both plants and animals. Yet, our knowledge about microRNA biogenesis and utilization in animals stems mostly from the study of Bilateria. In this study, we identified genes encoding the protein components of different parts of the microRNA pathway in Cnidaria, the likely sister phylum of Bilateria. These genes originated from three cnidarian lineages (sea anemones, stony corals, and hydras) that are separated by at least 500 My from one another. We studied the expression and phylogeny of the cnidarian homologs of Drosha and Pasha (DGCR8) that compose the microprocessor, the RNAse III enzyme Dicer and its partners, the HEN1 methyltransferase, the Argonaute protein effectors, as well as members of the GW182 protein family. We further reveal that whereas the bilaterian dicer partners Loquacious/TRBP and PACT are absent from Cnidaria, this phylum contains homologs of the double-stranded RNA-binding protein HYL1, the Dicer partner found in plants. We also identified HYL1 homologs in a sponge and a ctenophore. This finding raises questions regarding the independent evolution of the microRNA pathway in plants and animals, and together with the other results shed new light on the evolution of an important regulatory pathway. PMID:24030553

The evolution of microRNA pathway protein components in Cnidaria.

PubMed

Moran, Yehu; Praher, Daniela; Fredman, David; Technau, Ulrich

2013-12-01

In the last decade, it became evident that posttranscriptional regulation of gene expression by microRNAs is a central biological process in both plants and animals. Yet, our knowledge about microRNA biogenesis and utilization in animals stems mostly from the study of Bilateria. In this study, we identified genes encoding the protein components of different parts of the microRNA pathway in Cnidaria, the likely sister phylum of Bilateria. These genes originated from three cnidarian lineages (sea anemones, stony corals, and hydras) that are separated by at least 500 My from one another. We studied the expression and phylogeny of the cnidarian homologs of Drosha and Pasha (DGCR8) that compose the microprocessor, the RNAse III enzyme Dicer and its partners, the HEN1 methyltransferase, the Argonaute protein effectors, as well as members of the GW182 protein family. We further reveal that whereas the bilaterian dicer partners Loquacious/TRBP and PACT are absent from Cnidaria, this phylum contains homologs of the double-stranded RNA-binding protein HYL1, the Dicer partner found in plants. We also identified HYL1 homologs in a sponge and a ctenophore. This finding raises questions regarding the independent evolution of the microRNA pathway in plants and animals, and together with the other results shed new light on the evolution of an important regulatory pathway.

LABORATORY DIRECTED RESEARCH AND DEVELOPMENT ANNUAL REPORT TO THE DEPARTMENT OF ENERGY - DECEMBER 2006

DOE Office of Scientific and Technical Information (OSTI.GOV)

FOX, K.J.

Brookhaven National Laboratory (BNL) is a multidisciplinary laboratory that carries out basic and applied research in the physical, biomedical, and environmental sciences, and in selected energy technologies. It is managed by Brookhaven Science Associates, LLC, (BSA) under contract with the U. S. Department of Energy (DOE). BNL's total annual budget has averaged about $460 million. There are about 2,500 employees, and another 4,500 guest scientists and students who come each year to use the Laboratory's facilities and work with the staff. The BNL Laboratory Directed Research and Development (LDRD) Program reports its status to the U.S. Department of Energy (DOE)more » annually in March, as required by DOE Order 413.2B, ''Laboratory Directed Research and Development,'' April 19, 2006, and the Roles, Responsibilities, and Guidelines for Laboratory Directed Research and Development at the Department of Energy National Nuclear Security Administration Laboratories dated June 13, 2006. In accordance this is our Annual Report in which we describe the Purpose, Approach, Technical Progress and Results, and Specific Accomplishments of all LDRD projects that received funding during Fiscal Year 2006.« less

Doing that thing that scientists do: A discovery-driven module on protein purification and characterization for the undergraduate biochemistry laboratory classroom.

PubMed

Garrett, Teresa A; Osmundson, Joseph; Isaacson, Marisa; Herrera, Jennifer

2015-01-01

In traditional introductory biochemistry laboratory classes students learn techniques for protein purification and analysis by following provided, established, step-by-step procedures. Students are exposed to a variety of biochemical techniques but are often not developing procedures or collecting new, original data. In this laboratory module, students develop research skills through work on an original research project and gain confidence in their ability to design and execute an experiment while faculty can enhance their scholarly pursuits through the acquisition of original data in the classroom laboratory. Students are prepared for a 6-8 week discovery-driven project on the purification of the Escherichia coli cytidylate kinase (CMP kinase) through in class problems and other laboratory exercises on bioinformatics and protein structure analysis. After a minimal amount of guidance on how to perform the CMP kinase in vitro enzyme assay, SDS-PAGE, and the basics of protein purification, students, working in groups of three to four, develop a protein purification protocol based on the scientific literature and investigate some aspect of CMP kinase that interests them. Through this process, students learn how to implement a new but perhaps previously worked out procedure to answer their research question. In addition, they learn the importance of keeping a clear and thorough laboratory notebook and how to interpret their data and use that data to inform the next set of experiments. Following this module, students had increased confidence in their ability to do basic biochemistry techniques and reported that the "self-directed" nature of this lab increased their engagement in the project. © 2015 The International Union of Biochemistry and Molecular Biology.

Modeling the Evolution of Protein Domain Architectures Using Maximum Parsimony

PubMed Central

Fong, Jessica H.; Geer, Lewis Y.; Panchenko, Anna R.; Bryant, Stephen H.

2007-01-01

Domains are basic evolutionary units of proteins and most proteins have more than one domain. Advances in domain modeling and collection are making it possible to annotate a large fraction of known protein sequences by a linear ordering of their domains, yielding their architecture. Protein domain architectures link evolutionarily related proteins and underscore their shared functions. Here, we attempt to better understand this association by identifying the evolutionary pathways by which extant architectures may have evolved. We propose a model of evolution in which architectures arise through rearrangements of inferred precursor architectures and acquisition of new domains. These pathways are ranked using a parsimony principle, whereby scenarios requiring the fewest number of independent recombination events, namely fission and fusion operations, are assumed to be more likely. Using a data set of domain architectures present in 159 proteomes that represent all three major branches of the tree of life allows us to estimate the history of over 85% of all architectures in the sequence database. We find that the distribution of rearrangement classes is robust with respect to alternative parsimony rules for inferring the presence of precursor architectures in ancestral species. Analyzing the most parsimonious pathways, we find 87% of architectures to gain complexity over time through simple changes, among which fusion events account for 5.6 times as many architectures as fission. Our results may be used to compute domain architecture similarities, for example, based on the number of historical recombination events separating them. Domain architecture “neighbors” identified in this way may lead to new insights about the evolution of protein function. PMID:17166515

Collection Directions: The Evolution of Library Collections and Collecting

ERIC Educational Resources Information Center

Dempsey, Lorcan; Malpas, Constance; Lavoie, Brian

2014-01-01

This article takes a broad view of the evolution of collecting behaviors in a network environment and suggests some future directions based on various simple models. The authors look at the changing dynamics of print collections, at the greater engagement with research and learning behaviors, and at trends in scholarly communication. The goal is…

Laboratory evolution of copper tolerant yeast strains

PubMed Central

2012-01-01

Background Yeast strains endowed with robustness towards copper and/or enriched in intracellular Cu might find application in biotechnology processes, among others in the production of functional foods. Moreover, they can contribute to the study of human diseases related to impairments of copper metabolism. In this study, we investigated the molecular and physiological factors that confer copper tolerance to strains of baker's yeasts. Results We characterized the effects elicited in natural strains of Candida humilis and Saccharomyces cerevisiae by the exposure to copper in the culture broth. We observed that, whereas the growth of Saccharomyces cells was inhibited already at low Cu concentration, C. humilis was naturally robust and tolerated up to 1 g · L-1 CuSO4 in the medium. This resistant strain accumulated over 7 mg of Cu per gram of biomass and escaped severe oxidative stress thanks to high constitutive levels of superoxide dismutase and catalase. Both yeasts were then "evolved" to obtain hyper-resistant cells able to proliferate in high copper medium. While in S. cerevisiae the evolution of robustness towards Cu was paralleled by the increase of antioxidative enzymes, these same activities decreased in evolved hyper-resistant Candida cells. We also characterized in some detail changes in the profile of copper binding proteins, that appeared to be modified by evolution but, again, in a different way in the two yeasts. Conclusions Following evolution, both Candida and Saccharomyces cells were able to proliferate up to 2.5 g · L-1 CuSO4 and to accumulate high amounts of intracellular copper. The comparison of yeasts differing in their robustness, allowed highlighting physiological and molecular determinants of natural and acquired copper tolerance. We observed that different mechanisms contribute to confer metal tolerance: the control of copper uptake, changes in the levels of enzymes involved in oxidative stress response and changes in the copper

The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory

PubMed Central

Morris, Chris; Pajon, Anne; Griffiths, Susanne L.; Daniel, Ed; Savitsky, Marc; Lin, Bill; Diprose, Jonathan M.; Wilter da Silva, Alan; Pilicheva, Katya; Troshin, Peter; van Niekerk, Johannes; Isaacs, Neil; Naismith, James; Nave, Colin; Blake, Richard; Wilson, Keith S.; Stuart, David I.; Henrick, Kim; Esnouf, Robert M.

2011-01-01

The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service. PMID:21460443

The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory.

PubMed

Morris, Chris; Pajon, Anne; Griffiths, Susanne L; Daniel, Ed; Savitsky, Marc; Lin, Bill; Diprose, Jonathan M; da Silva, Alan Wilter; Pilicheva, Katya; Troshin, Peter; van Niekerk, Johannes; Isaacs, Neil; Naismith, James; Nave, Colin; Blake, Richard; Wilson, Keith S; Stuart, David I; Henrick, Kim; Esnouf, Robert M

2011-04-01

The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service.

Nucleotide exchange and excision technology DNA shuffling and directed evolution.

PubMed

Speck, Janina; Stebel, Sabine C; Arndt, Katja M; Müller, Kristian M

2011-01-01

Remarkable success in optimizing complex properties within DNA and proteins has been achieved by directed evolution. In contrast to various random mutagenesis methods and high-throughput selection methods, the number of available DNA shuffling procedures is limited, and protocols are often difficult to adjust. The strength of the nucleotide exchange and excision technology (NExT) DNA shuffling described here is the robust, efficient, and easily controllable DNA fragmentation step based on random incorporation of the so-called 'exchange nucleotides' by PCR. The exchange nucleotides are removed enzymatically, followed by chemical cleavage of the DNA backbone. The oligonucleotide pool is reassembled into full-length genes by internal primer extension, and the recombined gene library is amplified by standard PCR. The technique has been demonstrated by shuffling a defined gene library of chloramphenicol acetyltransferase variants using uridine as fragmentation defining exchange nucleotide. Substituting 33% of the dTTP with dUTP in the incorporation PCR resulted in shuffled clones with an average parental fragment size of 86 bases and revealed a mutation rate of only 0.1%. Additionally, a computer program (NExTProg) has been developed that predicts the fragment size distribution depending on the relative amount of the exchange nucleotide.

Retracing Evolution of Red Fluorescence in GFP-Like Proteins from Faviina Corals

PubMed Central

Field, Steven F.; Matz, Mikhail V.

2010-01-01

Proteins of the green fluorescent protein family represent a convenient experimental model to study evolution of novelty at the molecular level. Here, we focus on the origin of Kaede-like red fluorescent proteins characteristic of the corals of the Faviina suborder. We demonstrate, using an original approach involving resurrection and analysis of the library of possible evolutionary intermediates, that it takes on the order of 12 mutations, some of which strongly interact epistatically, to fully recapitulate the evolution of a red fluorescent phenotype from the ancestral green. Five of the identified mutations would not have been found without the help of ancestral reconstruction, because the corresponding site states are shared between extant red and green proteins due to their recent descent from a dual-function common ancestor. Seven of the 12 mutations affect residues that are not in close contact with the chromophore and thus must exert their effect indirectly through adjustments of the overall protein fold; the relevance of these mutations could not have been anticipated from the purely theoretical analysis of the protein's structure. Our results introduce a powerful experimental approach for comparative analysis of functional specificity in protein families even in the cases of pronounced epistasis, provide foundation for the detailed studies of evolutionary trajectories leading to novelty and complexity, and will help rational modification of existing fluorescent labels. PMID:19793832

Evol and ProDy for bridging protein sequence evolution and structural dynamics.

PubMed

Bakan, Ahmet; Dutta, Anindita; Mao, Wenzhi; Liu, Ying; Chennubhotla, Chakra; Lezon, Timothy R; Bahar, Ivet

2014-09-15

Correlations between sequence evolution and structural dynamics are of utmost importance in understanding the molecular mechanisms of function and their evolution. We have integrated Evol, a new package for fast and efficient comparative analysis of evolutionary patterns and conformational dynamics, into ProDy, a computational toolbox designed for inferring protein dynamics from experimental and theoretical data. Using information-theoretic approaches, Evol coanalyzes conservation and coevolution profiles extracted from multiple sequence alignments of protein families with their inferred dynamics. ProDy and Evol are open-source and freely available under MIT License from http://prody.csb.pitt.edu/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution

PubMed Central

Renata, Hans; Wang, Z. Jane

2015-01-01

High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe. PMID:25649694

Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins

NASA Astrophysics Data System (ADS)

Faure, Guilhem; Koonin, Eugene V.

2015-05-01

Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

Cerebrospinal fluid protein and glucose examinations and tuberculosis:
Will laboratory safety regulations force a change of practice?

PubMed

Tormey, William P; O'Hagan, Christopher

2015-01-01

Cerebrospinal fluid (CSF) protein and glucose examinations are usually performed in chemical pathology departments on autoanalysers. Tuberculosis (TB) is a group 3 biological agent under Directive 2000/54/EC of the European Parliament but in the biochemistry laboratory, no extra precautions are taken in its analysis in possible TB cases. The issue of laboratory practice and safety in the biochemical analyses of CSF specimens, when tuberculosis infection is in question is addressed in the context of ambiguity in the implementation of current national and international health and safety regulations. Additional protective measures for laboratory staff during the analysis of CSF TB samples should force a change in current laboratory practice and become a regulatory issue under ISO 15189. Annual Mantoux skin test or an interferon-γ release assay for TB should be mandatory for relevant staff. This manuscript addresses the issue of biochemistry laboratory practice and safety in the biochemical analyses of CSF specimens when tuberculosis infection is in question in the context of the ambiguity of statutory health and safety regulations.

Direct Calculation of Protein Fitness Landscapes through Computational Protein Design

PubMed Central

Au, Loretta; Green, David F.

2016-01-01

Naturally selected amino-acid sequences or experimentally derived ones are often the basis for understanding how protein three-dimensional conformation and function are determined by primary structure. Such sequences for a protein family comprise only a small fraction of all possible variants, however, representing the fitness landscape with limited scope. Explicitly sampling and characterizing alternative, unexplored protein sequences would directly identify fundamental reasons for sequence robustness (or variability), and we demonstrate that computational methods offer an efficient mechanism toward this end, on a large scale. The dead-end elimination and A∗ search algorithms were used here to find all low-energy single mutant variants, and corresponding structures of a G-protein heterotrimer, to measure changes in structural stability and binding interactions to define a protein fitness landscape. We established consistency between these algorithms with known biophysical and evolutionary trends for amino-acid substitutions, and could thus recapitulate known protein side-chain interactions and predict novel ones. PMID:26745411

Evolution of genitalia: theories, evidence, and new directions.

PubMed

Eberhard, William G

2010-01-01

Many hypotheses have been proposed to explain why male intromittent genitalia consistently tend to diverge more rapidly than other body traits of the same individuals in a wide range of animal taxa. Currently the two most popular involve sexual selection: sexually antagonistic coevolution (SAC) and cryptic female choice (CFC). A review of the most extensive attempts to discriminate between these two hypotheses indicates that SAC is not likely to have played a major role in explaining this pattern of genital evolution. Promising lines for future, more direct tests of CFC include experimental modification of male genital form and female sensory abilities, analysis of possible male-female dialogues during copulation, and direct observations of genital behavior.

The TIM Barrel Architecture Facilitated the Early Evolution of Protein-Mediated Metabolism.

PubMed

Goldman, Aaron David; Beatty, Joshua T; Landweber, Laura F

2016-01-01

The triosephosphate isomerase (TIM) barrel protein fold is a structurally repetitive architecture that is present in approximately 10% of all enzymes. It is generally assumed that this ubiquity in modern proteomes reflects an essential historical role in early protein-mediated metabolism. Here, we provide quantitative and comparative analyses to support several hypotheses about the early importance of the TIM barrel architecture. An information theoretical analysis of protein structures supports the hypothesis that the TIM barrel architecture could arise more easily by duplication and recombination compared to other mixed α/β structures. We show that TIM barrel enzymes corresponding to the most taxonomically broad superfamilies also have the broadest range of functions, often aided by metal and nucleotide-derived cofactors that are thought to reflect an earlier stage of metabolic evolution. By comparison to other putatively ancient protein architectures, we find that the functional diversity of TIM barrel proteins cannot be explained simply by their antiquity. Instead, the breadth of TIM barrel functions can be explained, in part, by the incorporation of a broad range of cofactors, a trend that does not appear to be shared by proteins in general. These results support the hypothesis that the simple and functionally general TIM barrel architecture may have arisen early in the evolution of protein biosynthesis and provided an ideal scaffold to facilitate the metabolic transition from ribozymes, peptides, and geochemical catalysts to modern protein enzymes.

Historical contingency and its biophysical basis in glucocorticoid receptor evolution.

PubMed

Harms, Michael J; Thornton, Joseph W

2014-08-14

Understanding how chance historical events shape evolutionary processes is a central goal of evolutionary biology. Direct insights into the extent and causes of evolutionary contingency have been limited to experimental systems, because it is difficult to know what happened in the deep past and to characterize other paths that evolution could have followed. Here we combine ancestral protein reconstruction, directed evolution and biophysical analysis to explore alternative 'might-have-been' trajectories during the ancient evolution of a novel protein function. We previously found that the evolution of cortisol specificity in the ancestral glucocorticoid receptor (GR) was contingent on permissive substitutions, which had no apparent effect on receptor function but were necessary for GR to tolerate the large-effect mutations that caused the shift in specificity. Here we show that alternative mutations that could have permitted the historical function-switching substitutions are extremely rare in the ensemble of genotypes accessible to the ancestral GR. In a library of thousands of variants of the ancestral protein, we recovered historical permissive substitutions but no alternative permissive genotypes. Using biophysical analysis, we found that permissive mutations must satisfy at least three physical requirements--they must stabilize specific local elements of the protein structure, maintain the correct energetic balance between functional conformations, and be compatible with the ancestral and derived structures--thus revealing why permissive mutations are rare. These findings demonstrate that GR evolution depended strongly on improbable, non-deterministic events, and this contingency arose from intrinsic biophysical properties of the protein.

Laboratory directed research and development fy1999 annual report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Ayat, R A

2000-04-11

The Lawrence Livermore National Laboratory (LLNL) was founded in 1952 and has been managed since its inception by the University of California (UC) for the U.S. Department of Energy (DOE). Because of this long association with UC, the Laboratory has been able to recruit a world-class workforce, establish an atmosphere of intellectual freedom and innovation, and achieve recognition in relevant fields of knowledge as a scientific and technological leader. This environment and reputation are essential for sustained scientific and technical excellence. As a DOE national laboratory with about 7,000 employees, LLNL has an essential and compelling primary mission to ensuremore » that the nation's nuclear weapons remain safe, secure, and reliable and to prevent the spread and use of nuclear weapons worldwide. The Laboratory receives funding from the DOE Assistant Secretary for Defense Programs, whose focus is stewardship of our nuclear weapons stockpile. Funding is also provided by the Deputy Administrator for Defense Nuclear Nonproliferation, many Department of Defense sponsors, other federal agencies, and the private sector. As a multidisciplinary laboratory, LLNL has applied its considerable skills in high-performance computing, advanced engineering, and the management of large research and development projects to become the science and technology leader in those areas of its mission responsibility. The Laboratory Directed Research and Development (LDRD) Program was authorized by the U.S. Congress in 1984. The Program allows the Director of each DOE laboratory to fund advanced, creative, and innovative research and development (R&D) activities that will ensure scientific and technical vitality in the continually evolving mission areas at DOE and the Laboratory. In addition, the LDRD Program provides LLNL with the flexibility to nurture and enrich essential scientific and technical competencies, which attract the most qualified scientists and engineers. The LDRD

Laboratory directed research and development program FY 1999

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, Todd; Levy, Karin

2000-03-08

The Ernest Orlando Lawrence Berkeley National Laboratory (Berkeley Lab or LBNL) is a multi-program national research facility operated by the University of California for the Department of Energy (DOE). As an integral element of DOE's National Laboratory System, Berkeley Lab supports DOE's missions in fundamental science, energy resources, and environmental quality. Berkeley Lab programs advance four distinct goals for DOE and the nation: (1) To perform leading multidisciplinary research in the computing sciences, physical sciences, energy sciences, biosciences, and general sciences in a manner that ensures employee and public safety and protection of the environment. (2) To develop and operatemore » unique national experimental facilities for qualified investigators. (3) To educate and train future generations of scientists and engineers to promote national science and education goals. (4) To transfer knowledge and technological innovations and to foster productive relationships among Berkeley Lab's research programs, universities, and industry in order to promote national economic competitiveness. This is the annual report on Laboratory Directed Research and Development (LDRD) program for FY99.« less

Small Laccase from "Streptomyces Coelicolor"--An Ideal Model Protein/Enzyme for Undergraduate Laboratory Experience

ERIC Educational Resources Information Center

Cook, Ryan; Hannon, Drew; Southard, Jonathan N.; Majumdar, Sudipta

2018-01-01

A one semester undergraduate biochemistry laboratory experience is described for an understanding of recombinant technology from gene cloning to protein characterization. An integrated experimental design includes three sequential modules: molecular cloning, protein expression and purification, and protein analysis and characterization. Students…

Did Convergent Protein Evolution Enable Phytoplasmas to Generate 'Zombie Plants'?

PubMed

Rümpler, Florian; Gramzow, Lydia; Theißen, Günter; Melzer, Rainer

2015-12-01

Phytoplasmas are pathogenic bacteria that reprogram plant development such that leaf-like structures instead of floral organs develop. Infected plants are sterile and mainly serve to propagate phytoplasmas and thus have been termed 'zombie plants'. The developmental reprogramming relies on specific interactions of the phytoplasma protein SAP54 with a small subset of MADS-domain transcription factors. Here, we propose that SAP54 folds into a structure that is similar to that of the K-domain, a protein-protein interaction domain of MADS-domain proteins. We suggest that undergoing convergent structural and sequence evolution, SAP54 evolved to mimic the K-domain. Given the high specificity of resulting developmental alterations, phytoplasmas might be used to study flower development in genetically intractable plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evolution and tinkering: what do a protein kinase, a transcriptional regulator and chromosome segregation/cell division proteins have in common?

PubMed

Derouiche, Abderahmane; Shi, Lei; Kalantari, Aida; Mijakovic, Ivan

2016-02-01

In this study, we focus on functional interactions among multi-domain proteins which share a common evolutionary origin. The examples we develop are four Bacillus subtilis proteins, which all possess an ATP-binding Walker motif: the bacterial tyrosine kinase (BY-kinase) PtkA, the chromosome segregation protein Soj (ParA), the cell division protein MinD and a transcription regulator SalA. These proteins have arisen via duplication of the ancestral ATP-binding domain, which has undergone fusions with other functional domains in the process of divergent evolution. We point out that these four proteins, despite having very different physiological roles, engage in an unusually high number of binary functional interactions. Namely, MinD attracts Soj and PtkA to the cell pole, and in addition, activates the kinase function of PtkA. SalA also activates the kinase function of PtkA, and it gets phosphorylated by PtkA as well. The consequence of this phosphorylation is the activation of SalA as a transcriptional repressor. We hypothesize that these functional interactions remain preserved during divergent evolution and represent a constraint on the process of evolutionary "tinkering", brought about by fusions of different functional domains.

Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell.

PubMed

Holthaus, Karin Brigit; Strasser, Bettina; Sipos, Wolfgang; Schmidt, Heiko A; Mlitz, Veronika; Sukseree, Supawadee; Weissenbacher, Anton; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

2016-03-01

The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Evolution of Mutual Mate Choice under Direct Benefits.

PubMed

Courtiol, Alexandre; Etienne, Loïc; Feron, Romain; Godelle, Bernard; Rousset, François

2016-11-01

In nature, the intensity of mate choice (i.e., choosiness) is highly variable within and between sexes. Despite growing empirical evidence of male and/or mutual mate choice, theoretical investigations of the joint evolution of female and male choosiness are few. In addition, previous approaches have often assumed an absence of trade-off between the direct benefits per mating and the lower mating rate that results from being choosy. Here we model the joint evolution of female and male choosiness when it is solely ruled by this fundamental trade-off. We show that this trade-off can generate a diversity of stable combinations of choosiness. Mutual mate choice can evolve only if both females and males exhibit long latency after mating. Furthermore, we show that an increase in choosiness in one sex does not necessarily prevent the evolution of mutual mate choice; the outcome depends on details shaping the trade-off: the life history, the decision rule for mate choice, and how the fecundity of a pair is shaped by the quality of both individuals. Last, we discuss the power of the sensitivity of the relative searching time (i.e., of the proportion of a lifetime spent searching for mates) as a predictor of the joint evolution of choosiness.

Direct detection of male quality can facilitate the evolution of female choosiness and indicators of good genes: Evolution across a continuum of indicator mechanisms.

PubMed

Dhole, Sumit; Stern, Caitlin A; Servedio, Maria R

2018-04-01

The evolution of mating displays as indicators of male quality has been the subject of extensive theoretical and empirical research for over four decades. Research has also addressed the evolution of female mate choice favoring such indicators. Yet, much debate still exists about whether displays can evolve through the indirect benefits of female mate choice. Here, we use a population genetic model to investigate how the extent to which females can directly detect male quality influences the evolution of female choosiness and male displays. We use a continuum framework that incorporates indicator mechanisms that are traditionally modeled separately. Counter to intuition, we find that intermediate levels of direct detection of male quality can facilitate, rather than impede, the evolution of female choosiness and male displays in broad regions of this continuum. We examine how this evolution is driven by selective forces on genetic quality and on the display, and find that direct detection of male quality results in stronger indirect selection favoring female choosiness. Our results imply that displays maybe more likely to evolve when female choosiness has already evolved to discriminate perceptible forms of male quality. They also highlight the importance of considering general female choosiness, as well as preference, in studies of "good genes." © 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.

Conservative and compensatory evolution in oxidative phosphorylation complexes of angiosperms with highly divergent rates of mitochondrial genome evolution.

PubMed

Havird, Justin C; Whitehill, Nicholas S; Snow, Christopher D; Sloan, Daniel B

2015-12-01

Interactions between nuclear and mitochondrial gene products are critical for eukaryotic cell function. Nuclear genes encoding mitochondrial-targeted proteins (N-mt genes) experience elevated rates of evolution, which has often been interpreted as evidence of nuclear compensation in response to elevated mitochondrial mutation rates. However, N-mt genes may be under relaxed functional constraints, which could also explain observed increases in their evolutionary rate. To disentangle these hypotheses, we examined patterns of sequence and structural evolution in nuclear- and mitochondrial-encoded oxidative phosphorylation proteins from species in the angiosperm genus Silene with vastly different mitochondrial mutation rates. We found correlated increases in N-mt gene evolution in species with fast-evolving mitochondrial DNA. Structural modeling revealed an overrepresentation of N-mt substitutions at positions that directly contact mutated residues in mitochondrial-encoded proteins, despite overall patterns of conservative structural evolution. These findings support the hypothesis that selection for compensatory changes in response to mitochondrial mutations contributes to the elevated rate of evolution in N-mt genes. We discuss these results in light of theories implicating mitochondrial mutation rates and mitonuclear coevolution as drivers of speciation and suggest comparative and experimental approaches that could take advantage of heterogeneity in rates of mtDNA evolution across eukaryotes to evaluate such theories. © 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.

Evolution of specifier proteins in glucosinolate-containing plants

PubMed Central

2012-01-01

Brassicaceae lineages as suggested by the phylogeny. The ability to form non-isothiocyanate products by specifier protein activity may provide plants with a selective advantage. The evolution of specifier proteins in the Brassicaceae demonstrates the plasticity of secondary metabolism within an activated plant defense system. PMID:22839361

Laboratory Calibration Studies in Support of ORGANICS on the International Space Station: Evolution of Organic Matter in Space

NASA Technical Reports Server (NTRS)

Ruiterkamp, R.; Ehrenfreund, P.; Halasinski, T.; Salama, F.; Foing, B.; Schmidt, W.

2002-01-01

This paper describes the scientific overview and current status of ORGANICS an exposure experiment performed on the International Space Station (ISS) to study the evolution of organic matter in space (PI: P. Ehrenfreund), with supporting laboratory experiments performed at NASA Ames. ORGANICS investigates the chemical evolution of samples submitted to long-duration exposure to space environment in near-Earth orbit. This experiment will provide information on the nature, evolution, and survival of carbon species in the interstellar medium (ISM) and in solar system targets.

The evolution of the protein synthesis system. I - A model of a primitive protein synthesis system

NASA Technical Reports Server (NTRS)

Mizutani, H.; Ponnamperuma, C.

1977-01-01

A model is developed to describe the evolution of the protein synthesis system. The model is comprised of two independent autocatalytic systems, one including one gene (A-gene) and two activated amino acid polymerases (O and A-polymerases), and the other including the addition of another gene (N-gene) and a nucleotide polymerase. Simulation results have suggested that even a small enzymic activity and polymerase specificity could lead the system to the most accurate protein synthesis, as far as permitted by transitions to systems with higher accuracy.

The direction of evolution: the rise of cooperative organization.

PubMed

Stewart, John E

2014-09-01

Two great trends are evident in the evolution of life on Earth: towards increasing diversification and towards increasing integration. Diversification has spread living processes across the planet, progressively increasing the range of environments and free energy sources exploited by life. Integration has proceeded through a stepwise process in which living entities at one level are integrated into cooperative groups that become larger-scale entities at the next level, and so on, producing cooperative organizations of increasing scale (for example, cooperative groups of simple cells gave rise to the more complex eukaryote cells, groups of these gave rise to multi-cellular organisms, and cooperative groups of these organisms produced animal societies). The trend towards increasing integration has continued during human evolution with the progressive increase in the scale of human groups and societies. The trends towards increasing diversification and integration are both driven by selection. An understanding of the trajectory and causal drivers of the trends suggests that they are likely to culminate in the emergence of a global entity. This entity would emerge from the integration of the living processes, matter, energy and technology of the planet into a global cooperative organization. Such an integration of the results of previous diversifications would enable the global entity to exploit the widest possible range of resources across the varied circumstances of the planet. This paper demonstrates that it's case for directionality meets the tests and criticisms that have proven fatal to previous claims for directionality in evolution. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Predicting functional divergence in protein evolution by site-specific rate shifts

NASA Technical Reports Server (NTRS)

Gaucher, Eric A.; Gu, Xun; Miyamoto, Michael M.; Benner, Steven A.

2002-01-01

Most modern tools that analyze protein evolution allow individual sites to mutate at constant rates over the history of the protein family. However, Walter Fitch observed in the 1970s that, if a protein changes its function, the mutability of individual sites might also change. This observation is captured in the "non-homogeneous gamma model", which extracts functional information from gene families by examining the different rates at which individual sites evolve. This model has recently been coupled with structural and molecular biology to identify sites that are likely to be involved in changing function within the gene family. Applying this to multiple gene families highlights the widespread divergence of functional behavior among proteins to generate paralogs and orthologs.

Protein structure and evolution: are they constrained globally by a principle derived from information theory?

PubMed

Hatton, Leslie; Warr, Gregory

2015-01-01

That the physicochemical properties of amino acids constrain the structure, function and evolution of proteins is not in doubt. However, principles derived from information theory may also set bounds on the structure (and thus also the evolution) of proteins. Here we analyze the global properties of the full set of proteins in release 13-11 of the SwissProt database, showing by experimental test of predictions from information theory that their collective structure exhibits properties that are consistent with their being guided by a conservation principle. This principle (Conservation of Information) defines the global properties of systems composed of discrete components each of which is in turn assembled from discrete smaller pieces. In the system of proteins, each protein is a component, and each protein is assembled from amino acids. Central to this principle is the inter-relationship of the unique amino acid count and total length of a protein and its implications for both average protein length and occurrence of proteins with specific unique amino acid counts. The unique amino acid count is simply the number of distinct amino acids (including those that are post-translationally modified) that occur in a protein, and is independent of the number of times that the particular amino acid occurs in the sequence. Conservation of Information does not operate at the local level (it is independent of the physicochemical properties of the amino acids) where the influences of natural selection are manifest in the variety of protein structure and function that is well understood. Rather, this analysis implies that Conservation of Information would define the global bounds within which the whole system of proteins is constrained; thus it appears to be acting to constrain evolution at a level different from natural selection, a conclusion that appears counter-intuitive but is supported by the studies described herein.

Directed energy deflection laboratory measurements

NASA Astrophysics Data System (ADS)

Brashears, Travis; Lubin, Phillip; Hughes, Gary B.; Meinhold, Peter; Suen, Jonathan; Batliner, Payton; Motta, Caio; Griswold, Janelle; Kangas, Miikka; Johansson, Isbella; Alnawakhtha, Yusuf; Prater, Kenyon; Lang, Alex; Madajian, Jonathan

2015-09-01

We report on laboratory studies of the effectiveness of directed energy planetary defense as a part of the DESTAR (Directed Energy System for Targeting of Asteroids and exploRation) program. DE-STAR [1][5][6] and DE-STARLITE [2][5][6] are directed energy "stand-off" and "stand-on" programs, respectively. These systems consist of a modular array of kilowatt-class lasers powered by photovoltaics, and are capable of heating a spot on the surface of an asteroid to the point of vaporization. Mass ejection, as a plume of evaporated material, creates a reactionary thrust capable of diverting the asteroid's orbit. In a series of papers, we have developed a theoretical basis and described numerical simulations for determining the thrust produced by material evaporating from the surface of an asteroid [1][2][3][4][5][6]. In the DE-STAR concept, the asteroid itself is used as the deflection "propellant". This study presents results of experiments designed to measure the thrust created by evaporation from a laser directed energy spot. We constructed a vacuum chamber to simulate space conditions, and installed a torsion balance that holds an "asteroid" sample. The sample is illuminated with a fiber array laser with flux levels up to 60 MW/m2 which allows us to simulate a mission level flux but on a small scale. We use a separate laser as well as a position sensitive centroid detector to readout the angular motion of the torsion balance and can thus determine the thrust. We compare the measured thrust to the models. Our theoretical models indicate a coupling coefficient well in excess of 100 μN/Woptical, though we assume a more conservative value of 80 μN/Woptical and then degrade this with an optical "encircled energy" efficiency of 0.75 to 60 μN/Woptical in our deflection modeling. Our measurements discussed here yield about 45 μN/Wabsorbed as a reasonable lower limit to the thrust per optical watt absorbed.

A chemical biology approach demonstrates G protein βγ subunits are sufficient to mediate directional neutrophil chemotaxis.

PubMed

Surve, Chinmay R; Lehmann, David; Smrcka, Alan V

2014-06-20

Our laboratory has identified a number of small molecules that bind to G protein βγ subunits (Gβγ) by competing for peptide binding to the Gβγ "hot spot." M119/Gallein were identified as inhibitors of Gβγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on Gβγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γ activation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca(2+) release, and activated other downstream targets of Gβγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases Gβγ in vitro from Gαi1β1γ2 heterotrimers by causing its dissociation from GαGDP without inducing nucleotide exchange in the Gα subunit. We used this novel probe to examine the hypothesis that Gβγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free Gβγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Rab protein evolution and the history of the eukaryotic endomembrane system

PubMed Central

Brighouse, Andrew; Dacks, Joel B.

2010-01-01

Spectacular increases in the quantity of sequence data genome have facilitated major advances in eukaryotic comparative genomics. By exploiting homology with classical model organisms, this makes possible predictions of pathways and cellular functions currently impossible to address in intractable organisms. Echoing realization that core metabolic processes were established very early following evolution of life on earth, it is now emerging that many eukaryotic cellular features, including the endomembrane system, are ancient and organized around near-universal principles. Rab proteins are key mediators of vesicle transport and specificity, and via the presence of multiple paralogues, alterations in interaction specificity and modification of pathways, contribute greatly to the evolution of complexity of membrane transport. Understanding system-level contributions of Rab proteins to evolutionary history provides insight into the multiple processes sculpting cellular transport pathways and the exciting challenges that we face in delving further into the origins of membrane trafficking specificity. PMID:20582450

Green Fluorescent Protein-Focused Bioinformatics Laboratory Experiment Suitable for Undergraduates in Biochemistry Courses

ERIC Educational Resources Information Center

Rowe, Laura

2017-01-01

An introductory bioinformatics laboratory experiment focused on protein analysis has been developed that is suitable for undergraduate students in introductory biochemistry courses. The laboratory experiment is designed to be potentially used as a "stand-alone" activity in which students are introduced to basic bioinformatics tools and…

Binding Direction-Based Two-Dimensional Flattened Contact Area Computing Algorithm for Protein-Protein Interactions.

PubMed

Kang, Beom Sik; Pugalendhi, GaneshKumar; Kim, Ku-Jin

2017-10-13

Interactions between protein molecules are essential for the assembly, function, and regulation of proteins. The contact region between two protein molecules in a protein complex is usually complementary in shape for both molecules and the area of the contact region can be used to estimate the binding strength between two molecules. Although the area is a value calculated from the three-dimensional surface, it cannot represent the three-dimensional shape of the surface. Therefore, we propose an original concept of two-dimensional contact area which provides further information such as the ruggedness of the contact region. We present a novel algorithm for calculating the binding direction between two molecules in a protein complex, and then suggest a method to compute the two-dimensional flattened area of the contact region between two molecules based on the binding direction.

Lab-on-a-chip in vitro compartmentalization technologies for protein studies.

PubMed

Zhu, Yonggang; Power, Barbara E

2008-01-01

In vitro compartmentalization (IVC) is a powerful tool for studying protein-protein reactions, due to its high capacity and the versatility of droplet technologies. IVC bridges the gap between chemistry and biology as it enables the incorporation of unnatural amino acids with modifications into biological systems, through protein transcription and translation reactions, in a cell-like microdrop environment. The quest for the ultimate chip for protein studies using IVC is the drive for the development of various microfluidic droplet technologies to enable these unusual biochemical reactions to occur. These techniques have been shown to generate precise microdrops with a controlled size. Various chemical and physical phenomena have been utilized for on-chip manipulation to allow the droplets to be generated, fused, and split. Coupled with detection techniques, droplets can be sorted and selected. These capabilities allow directed protein evolution to be carried out on a microchip. With further technological development of the detection module, factors such as addressable storage, transport and interfacing technologies, could be integrated and thus provide platforms for protein studies with high efficiency and accuracy that conventional laboratories cannot achieve.

BRCA1 interacts directly with the Fanconi anemia protein FANCA.

PubMed

Folias, Alexandra; Matkovic, Mara; Bruun, Donald; Reid, Sonja; Hejna, James; Grompe, Markus; D'Andrea, Alan; Moses, Robb

2002-10-01

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by skeletal defects, anemia, chromosomal instability and increased risk of leukemia. At the cellular level FA is characterized by increased sensitivity to agents forming interstrand crosslinks (ICL) in DNA. Six FA genes have been cloned and interactions among individual FANC proteins have been found. The FANCD2 protein co-localizes in nuclear foci with the BRCA1 protein following DNA damage and during S-phase, requiring the FANCA, C, E and G proteins to do so. This finding may reflect a direct role for the BRCA1 protein in double strand break (DSB) repair and interaction with the FANC proteins. Therefore interactions between BRCA1 and the FANC proteins were investigated. Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740-1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.

Evolution and functional divergence of the anoctamin family of membrane proteins

PubMed Central

2010-01-01

Background The anoctamin family of transmembrane proteins are found in all eukaryotes and consists of 10 members in vertebrates. Ano1 and ano2 were observed to have Ca2+ activated Cl- channel activity. Recent findings however have revealed that ano6, and ano7 can also produce chloride currents, although with different properties. In contrast, ano9 and ano10 suppress baseline Cl- conductance when co-expressed with ano1 thus suggesting that different anoctamins can interfere with each other. In order to elucidate intrinsic functional diversity, and underlying evolutionary mechanism among anoctamins, we performed comprehensive bioinformatics analysis of anoctamin gene family. Results Our results show that anoctamin protein paralogs evolved from several gene duplication events followed by functional divergence of vertebrate anoctamins. Most of the amino acid replacements responsible for the functional divergence were fixed by adaptive evolution and this seem to be a common pattern in anoctamin gene family evolution. Strong purifying selection and the loss of many gene duplication products indicate rigid structure-function relationships among anoctamins. Conclusions Our study suggests that anoctamins have evolved by series of duplication events, and that they are constrained by purifying selection. In addition we identified a number of protein domains, and amino acid residues which contribute to predicted functional divergence. Hopefully, this work will facilitate future functional characterization of the anoctamin membrane protein family. PMID:20964844

Identification of novel direct protein-protein interactions by irradiating living cells with femtosecond UV laser pulses.

PubMed

Itri, Francesco; Monti, Daria Maria; Chino, Marco; Vinciguerra, Roberto; Altucci, Carlo; Lombardi, Angela; Piccoli, Renata; Birolo, Leila; Arciello, Angela

2017-10-07

The identification of protein-protein interaction networks in living cells is becoming increasingly fundamental to elucidate main biological processes and to understand disease molecular bases on a system-wide level. We recently described a method (LUCK, Laser UV Cross-linKing) to cross-link interacting protein surfaces in living cells by UV laser irradiation. By using this innovative methodology, that does not require any protein modification or cell engineering, here we demonstrate that, upon UV laser irradiation of HeLa cells, a direct interaction between GAPDH and alpha-enolase was "frozen" by a cross-linking event. We validated the occurrence of this direct interaction by co-immunoprecipitation and Immuno-FRET analyses. This represents a proof of principle of the LUCK capability to reveal direct protein interactions in their physiological environment. Copyright © 2017 Elsevier Inc. All rights reserved.

Varying iron release from transferrin and lactoferrin proteins. A laboratory experiment.

PubMed

Carmona, Fernando; González, Ana; Sánchez, Manu; Gálvez, Natividad; Cuesta, Rafael; Capdevila, Mercè; Dominguez-Vera, Jose M

2017-11-01

Iron metabolism is an important subject of study for undergraduate students of chemistry and biochemistry. Relevant laboratory exercises are scarce in the literature but would be very helpful in assisting students grasp key concepts. The experiment described here deals with different iron release mechanisms of two protagonists in iron metabolism: serum transferrin (Tf) and lactoferrin (Lf). Despite having very similar structures and iron-binding sites, Tf releases practically all its iron at pH 5.5 while Lf requires a significantly lower pH of 3. This difference in behavior is directly related to their respective biological functions as Tf blood-borne iron into the cell, while Lf competes with pathogens to sequester iron in biological fluids at more acidic pHs.  During this experiment, the students will carry out iron loading and unloading on both human Lf and Tf and monitor the iron release at different pHs using UV-Vis spectroscopy. With this simple approach, the students will discover the different patterns of iron release of Tf and Lf and how this variance in behavior relates to their biological functions. Furthermore, this laboratory practice can be expanded to allow students to investigate a variety of iron proteins. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):521-527, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

PubMed

Thinon, Emmanuelle; Fernandez, Joseph P; Molina, Henrik; Hang, Howard C

2018-05-04

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

Dynamic New World: Refining Our View of Protein Structure, Function and Evolution

PubMed Central

Mannige, Ranjan V.

2014-01-01

Proteins are crucial to the functioning of all lifeforms. Traditional understanding posits that a single protein occupies a single structure (“fold”), which performs a single function. This view is radically challenged with the recognition that high structural dynamism—the capacity to be extra “floppy”—is more prevalent in functional proteins than previously assumed. As reviewed here, this dynamic take on proteins affects our understanding of protein “structure”, function, and evolution, and even gives us a glimpse into protein origination. Specifically, this review will discuss historical developments concerning protein structure, and important new relationships between dynamism and aspects of protein sequence, structure, binding modes, binding promiscuity, evolvability, and origination. Along the way, suggestions will be provided for how key parts of textbook definitions—that so far have excluded membership to intrinsically disordered proteins (IDPs)—could be modified to accommodate our more dynamic understanding of proteins. PMID:28250374

An Adaptable Investigative Graduate Laboratory Course for Teaching Protein Purification

ERIC Educational Resources Information Center

Carroll, Christopher W.; Keller, Lani C.

2014-01-01

This adaptable graduate laboratory course on protein purification offers students the opportunity to explore a wide range of techniques while allowing the instructor the freedom to incorporate their own personal research interests. The course design involves two sequential purification schemes performed in a single semester. The first part…

The TALE face of Hox proteins in animal evolution.

PubMed

Merabet, Samir; Galliot, Brigitte

2015-01-01

Hox genes are major regulators of embryonic development. One of their most conserved functions is to coordinate the formation of specific body structures along the anterior-posterior (AP) axis in Bilateria. This architectural role was at the basis of several morphological innovations across bilaterian evolution. In this review, we traced the origin of the Hox patterning system by considering the partnership with PBC and Meis proteins. PBC and Meis belong to the TALE-class of homeodomain-containing transcription factors and act as generic cofactors of Hox proteins for AP axis patterning in Bilateria. Recent data indicate that Hox proteins acquired the ability to interact with their TALE partners in the last common ancestor of Bilateria and Cnidaria. These interactions relied initially on a short peptide motif called hexapeptide (HX), which is present in Hox and non-Hox protein families. Remarkably, Hox proteins can also recruit the TALE cofactors by using specific PBC Interaction Motifs (SPIMs). We describe how a functional Hox/TALE patterning system emerged in eumetazoans through the acquisition of SPIMs. We anticipate that interaction flexibility could be found in other patterning systems, being at the heart of the astonishing morphological diversity observed in the animal kingdom.

Laboratory directed research and development program FY 1997

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1998-03-01

This report compiles the annual reports of Laboratory Directed Research and Development projects supported by the Berkeley Lab. Projects are arranged under the following topical sections: (1) Accelerator and fusion research division; (2) Chemical sciences division; (3) Computing Sciences; (4) Earth sciences division; (5) Environmental energy technologies division; (6) life sciences division; (7) Materials sciences division; (8) Nuclear science division; (9) Physics division; (10) Structural biology division; and (11) Cross-divisional. A total of 66 projects are summarized.

Laboratory information management system for membrane protein structure initiative--from gene to crystal.

PubMed

Troshin, Petr V; Morris, Chris; Prince, Stephen M; Papiz, Miroslav Z

2008-12-01

Membrane Protein Structure Initiative (MPSI) exploits laboratory competencies to work collaboratively and distribute work among the different sites. This is possible as protein structure determination requires a series of steps, starting with target selection, through cloning, expression, purification, crystallization and finally structure determination. Distributed sites create a unique set of challenges for integrating and passing on information on the progress of targets. This role is played by the Protein Information Management System (PIMS), which is a laboratory information management system (LIMS), serving as a hub for MPSI, allowing collaborative structural proteomics to be carried out in a distributed fashion. It holds key information on the progress of cloning, expression, purification and crystallization of proteins. PIMS is employed to track the status of protein targets and to manage constructs, primers, experiments, protocols, sample locations and their detailed histories: thus playing a key role in MPSI data exchange. It also serves as the centre of a federation of interoperable information resources such as local laboratory information systems and international archival resources, like PDB or NCBI. During the challenging task of PIMS integration, within the MPSI, we discovered a number of prerequisites for successful PIMS integration. In this article we share our experiences and provide invaluable insights into the process of LIMS adaptation. This information should be of interest to partners who are thinking about using LIMS as a data centre for their collaborative efforts.

Mate Choice in Soldier Beetles: Field & Laboratory Experiments that Demonstrate Sexual Selection in Action

ERIC Educational Resources Information Center

Eason, Perri K.; Sherman, Peter T.

2003-01-01

Although the theory of evolution is the foundation of modern biology, students too rarely have an opportunity to watch selection operate in natural populations of animals. This lack may be partially responsible for the unfortunate ignorance of many people regarding the significance of evolution in biology. Laboratory exercises that directly study…

Exploring Hydrogen Evolution and the Overpotential

ERIC Educational Resources Information Center

Lyon, Yana A.; Roberts, Adrienne A.; McMillin, David R.

2015-01-01

The laboratory experiment described provides insight into the energetics of hydrogen evolution at an electrode as well as the intrinsic barrier that typically impedes reaction. In the course of the exercise, students find that Sn(s) is thermodynamically capable of combining with protons to form hydrogen, but that the direct reaction occurs at a…

Construction and Potential Applications of Biosensors for Proteins in Clinical Laboratory Diagnosis

PubMed Central

Liu, Xuan

2017-01-01

Biosensors for proteins have shown attractive advantages compared to traditional techniques in clinical laboratory diagnosis. In virtue of modern fabrication modes and detection techniques, various immunosensing platforms have been reported on basis of the specific recognition between antigen-antibody pairs. In addition to profit from the development of nanotechnology and molecular biology, diverse fabrication and signal amplification strategies have been designed for detection of protein antigens, which has led to great achievements in fast quantitative and simultaneous testing with extremely high sensitivity and specificity. Besides antigens, determination of antibodies also possesses great significance for clinical laboratory diagnosis. In this review, we will categorize recent immunosensors for proteins by different detection techniques. The basic conception of detection techniques, sensing mechanisms, and the relevant signal amplification strategies are introduced. Since antibodies and antigens have an equal position to each other in immunosensing, all biosensing strategies for antigens can be extended to antibodies under appropriate optimizations. Biosensors for antibodies are summarized, focusing on potential applications in clinical laboratory diagnosis, such as a series of biomarkers for infectious diseases and autoimmune diseases, and an evaluation of vaccine immunity. The excellent performances of these biosensors provide a prospective space for future antibody-detection-based disease serodiagnosis. PMID:29207528

Construction and Potential Applications of Biosensors for Proteins in Clinical Laboratory Diagnosis.

PubMed

Liu, Xuan; Jiang, Hui

2017-12-04

Biosensors for proteins have shown attractive advantages compared to traditional techniques in clinical laboratory diagnosis. In virtue of modern fabrication modes and detection techniques, various immunosensing platforms have been reported on basis of the specific recognition between antigen-antibody pairs. In addition to profit from the development of nanotechnology and molecular biology, diverse fabrication and signal amplification strategies have been designed for detection of protein antigens, which has led to great achievements in fast quantitative and simultaneous testing with extremely high sensitivity and specificity. Besides antigens, determination of antibodies also possesses great significance for clinical laboratory diagnosis. In this review, we will categorize recent immunosensors for proteins by different detection techniques. The basic conception of detection techniques, sensing mechanisms, and the relevant signal amplification strategies are introduced. Since antibodies and antigens have an equal position to each other in immunosensing, all biosensing strategies for antigens can be extended to antibodies under appropriate optimizations. Biosensors for antibodies are summarized, focusing on potential applications in clinical laboratory diagnosis, such as a series of biomarkers for infectious diseases and autoimmune diseases, and an evaluation of vaccine immunity. The excellent performances of these biosensors provide a prospective space for future antibody-detection-based disease serodiagnosis.

Directed self-assembly of proteins into discrete radial patterns

PubMed Central

Thakur, Garima; Prashanthi, Kovur; Thundat, Thomas

2013-01-01

Unlike physical patterning of materials at nanometer scale, manipulating soft matter such as biomolecules into patterns is still in its infancy. Self-assembled monolayer (SAM) with surface density gradient has the capability to drive biomolecules in specific directions to create hierarchical and discrete structures. Here, we report on a two-step process of self-assembly of the human serum albumin (HSA) protein into discrete ring structures based on density gradient of SAM. The methodology involves first creating a 2-dimensional (2D) polyethylene glycol (PEG) islands with responsive carboxyl functionalities. Incubation of proteins on such pre-patterned surfaces results in direct self-assembly of protein molecules around PEG islands. Immobilization and adsorption of protein on such structures over time evolve into the self-assembled patterns. PMID:23719678

Fluorescence Quantum Yield Measurements of Fluorescent Proteins: A Laboratory Experiment for a Biochemistry or Molecular Biophysics Laboratory Course

ERIC Educational Resources Information Center

Wall, Kathryn P.; Dillon, Rebecca; Knowles, Michelle K.

2015-01-01

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts…

Acquisition of Triacylglycerol Transfer Activity by Microsomal Triglyceride Transfer Protein During Evolution

PubMed Central

Rava, Paul; Hussain, M. Mahmood

2008-01-01

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of neutral lipid rich apolipoprotein B (apoB)-lipoproteins. Previously we reported that the Drosophila MTP transfers phospholipids but does not transfer triglycerides. In contrast, human MTP transfers both lipids. To explore the acquisition of triglyceride transfer activity by MTP, we evaluated amino acid sequences, protein structures, as well as the biochemical and cellular properties of various MTP orthologs obtained from species that diverged during evolution. All MTP orthologs shared similar secondary and tertiary structures, associated with protein disulfide isomerase, localized to the endoplasmic reticulum, and supported apoB secretion. While vertebrate MTPs transferred triglyceride invertebrate MTPs lacked this activity. Thus, triglyceride transfer activity was acquired during the transition from invertebrates to vertebrates. Within vertebrates, fish, amphibians, and birds displayed 27%, 40% and 100% triglyceride transfer activity compared to mammals. We conclude that MTP triglyceride transfer activity first appeared in fish and speculate that the acquisition of triglyceride transfer activity by MTP provided for a significant advantage in the evolution of larger and more complex organisms. PMID:17924655

PROFESS: a PROtein Function, Evolution, Structure and Sequence database

PubMed Central

Triplet, Thomas; Shortridge, Matthew D.; Griep, Mark A.; Stark, Jaime L.; Powers, Robert; Revesz, Peter

2010-01-01

The proliferation of biological databases and the easy access enabled by the Internet is having a beneficial impact on biological sciences and transforming the way research is conducted. There are ∼1100 molecular biology databases dispersed throughout the Internet. To assist in the functional, structural and evolutionary analysis of the abundant number of novel proteins continually identified from whole-genome sequencing, we introduce the PROFESS (PROtein Function, Evolution, Structure and Sequence) database. Our database is designed to be versatile and expandable and will not confine analysis to a pre-existing set of data relationships. A fundamental component of this approach is the development of an intuitive query system that incorporates a variety of similarity functions capable of generating data relationships not conceived during the creation of the database. The utility of PROFESS is demonstrated by the analysis of the structural drift of homologous proteins and the identification of potential pancreatic cancer therapeutic targets based on the observation of protein–protein interaction networks. Database URL: http://cse.unl.edu/∼profess/ PMID:20624718

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

PubMed Central

Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P.

2016-01-01

Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

Laboratory directed research and development. FY 1995 progress report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vigil, J.; Prono, J.

1996-03-01

This document presents an overview of Laboratory Directed Research and Development Programs at Los Alamos. The nine technical disciplines in which research is described include materials, engineering and base technologies, plasma, fluids, and particle beams, chemistry, mathematics and computational science, atmic and molecular physics, geoscience, space science, and astrophysics, nuclear and particle physics, and biosciences. Brief descriptions are provided in the above programs.

LBNL Laboratory Directed Research and Development Program FY2016

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, D.

2017-03-01

The Berkeley Lab Laboratory Directed Research and Development Program FY2016 report is compiled from annual reports submitted by principal investigators following the close of the fiscal year. This report describes the supported projects and summarizes their accomplishments. It constitutes a part of the LDRD program planning and documentation process that includes an annual planning cycle, project selection, implementation and review.

Combining protein sequence, structure, and dynamics: A novel approach for functional evolution analysis of PAS domain superfamily.

PubMed

Dong, Zheng; Zhou, Hongyu; Tao, Peng

2018-02-01

PAS domains are widespread in archaea, bacteria, and eukaryota, and play important roles in various functions. In this study, we aim to explore functional evolutionary relationship among proteins in the PAS domain superfamily in view of the sequence-structure-dynamics-function relationship. We collected protein sequences and crystal structure data from RCSB Protein Data Bank of the PAS domain superfamily belonging to three biological functions (nucleotide binding, photoreceptor activity, and transferase activity). Protein sequences were aligned and then used to select sequence-conserved residues and build phylogenetic tree. Three-dimensional structure alignment was also applied to obtain structure-conserved residues. The protein dynamics were analyzed using elastic network model (ENM) and validated by molecular dynamics (MD) simulation. The result showed that the proteins with same function could be grouped by sequence similarity, and proteins in different functional groups displayed statistically significant difference in their vibrational patterns. Interestingly, in all three functional groups, conserved amino acid residues identified by sequence and structure conservation analysis generally have a lower fluctuation than other residues. In addition, the fluctuation of conserved residues in each biological function group was strongly correlated with the corresponding biological function. This research suggested a direct connection in which the protein sequences were related to various functions through structural dynamics. This is a new attempt to delineate functional evolution of proteins using the integrated information of sequence, structure, and dynamics. © 2017 The Protein Society.

Zebra: a web server for bioinformatic analysis of diverse protein families.

PubMed

Suplatov, Dmitry; Kirilin, Evgeny; Takhaveev, Vakil; Svedas, Vytas

2014-01-01

During evolution of proteins from a common ancestor, one functional property can be preserved while others can vary leading to functional diversity. A systematic study of the corresponding adaptive mutations provides a key to one of the most challenging problems of modern structural biology - understanding the impact of amino acid substitutions on protein function. The subfamily-specific positions (SSPs) are conserved within functional subfamilies but are different between them and, therefore, seem to be responsible for functional diversity in protein superfamilies. Consequently, a corresponding method to perform the bioinformatic analysis of sequence and structural data has to be implemented in the common laboratory practice to study the structure-function relationship in proteins and develop novel protein engineering strategies. This paper describes Zebra web server - a powerful remote platform that implements a novel bioinformatic analysis algorithm to study diverse protein families. It is the first application that provides specificity determinants at different levels of functional classification, therefore addressing complex functional diversity of large superfamilies. Statistical analysis is implemented to automatically select a set of highly significant SSPs to be used as hotspots for directed evolution or rational design experiments and analyzed studying the structure-function relationship. Zebra results are provided in two ways - (1) as a single all-in-one parsable text file and (2) as PyMol sessions with structural representation of SSPs. Zebra web server is available at http://biokinet.belozersky.msu.ru/zebra .

Laboratory-Directed Research and Development 2016 Summary Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pillai, Rekha Sukamar; Jacobson, Julie Ann

The Laboratory-Directed Research and Development (LDRD) Program at Idaho National Laboratory (INL) reports its status to the U.S. Department of Energy (DOE) by March of each year. The program operates under the authority of DOE Order 413.2C, “Laboratory Directed Research and Development” (April 19, 2006), which establishes DOE’s requirements for the program while providing the laboratory director broad flexibility for program implementation. LDRD funds are obtained through a charge to all INL programs. This report includes summaries of all INL LDRD research activities supported during Fiscal Year (FY) 2016. INL is the lead laboratory for the DOE Office of Nuclearmore » Energy (DOE-NE). The INL mission is to discover, demonstrate, and secure innovative nuclear energy solutions, other clean energy options, and critical infrastructure with a vision to change the world’s energy future and secure our critical infrastructure. Operating since 1949, INL is the nation’s leading research, development, and demonstration center for nuclear energy, including nuclear nonproliferation and physical and cyber-based protection of energy systems and critical infrastructure, as well as integrated energy systems research, development, demonstration, and deployment. INL has been managed and operated by Battelle Energy Alliance, LLC (a wholly owned company of Battelle) for DOE since 2005. Battelle Energy Alliance, LLC, is a partnership between Battelle, BWX Technologies, Inc., AECOM, the Electric Power Research Institute, the National University Consortium (Massachusetts Institute of Technology, Ohio State University, North Carolina State University, University of New Mexico, and Oregon State University), and the Idaho university collaborators (i.e., University of Idaho, Idaho State University, and Boise State University). Since its creation, INL’s research and development (R&D) portfolio has broadened with targeted programs supporting national missions to advance nuclear

Laboratory Directed Research & Development program. Annual report to the Department of Energy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogeka, G.J.; Romano, A.J.

1995-12-01

This report briefly discusses the following projects coordinated at Brookhaven National Laboratory: investigation of the utility of max-entropy methods for the analysis of powder diffraction data; analysis of structures and interactions of nucleic acids and proteins by small angle x-ray diffraction; relaxographic MRI and functional MRI; very low temperature infra-red laser absorption as a potential analytical tool; state-resolved measurements of H{sub 2} photodesorption: development of laser probes of H{sub 2} for in-situ accelerator measurements; Siberian snake prototype development for RHIC; synthesis and characterization of novel microporous solids; ozone depletion, chemistry and physics of stratospheric aerosols; understanding the molecular basis formore » the synthesis of plant fatty acids possessing unusual double bond positions; structure determination of outer surface proteins of the Lyme disease spirochete; low mass, low-cost multi-wire proportional chambers for muon systems of collider experiments; theory of self-organized criticality; development of the PCR-SSCP technique for the detection, at the single cell level, of specific genetic changes; feasibility of SPECT in imaging of F-18 FDG accumulation in tumors; visible free electron laser oscillator experiment; study of possible 2 + 2 TeV muon-muon collider; ultraviolet FEL R & D; precision machining using hard x-rays; new directions in in-vivo enzyme mapping: catechol-O-methyltransferase; proposal to develop a high rate muon polarimeter; development of intense, tunable 20-femtosecond laser systems; use of extreme thermophilic bacterium thermatoga maritima as a source of ribosomal components and translation factors for structural studies; and biochemical and structural studies of Chaperon proteins from thermophilic bacteria and other experiments.« less

Argonne National Laboratory annual report of Laboratory Directed Research and Development Program Activities FY 2009.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Office of the Director

2010-04-09

I am pleased to submit Argonne National Laboratory's Annual Report on its Laboratory Directed Research and Development (LDRD) activities for fiscal year 2009. Fiscal year 2009 saw a heightened focus by DOE and the nation on the need to develop new sources of energy. Argonne scientists are investigating many different sources of energy, including nuclear, solar, and biofuels, as well as ways to store, use, and transmit energy more safely, cleanly, and efficiently. DOE selected Argonne as the site for two new Energy Frontier Research Centers (EFRCs) - the Institute for Atom-Efficient Chemical Transformations and the Center for Electrical Energymore » Storage - and funded two other EFRCs to which Argonne is a major partner. The award of at least two of the EFRCs can be directly linked to early LDRD-funded efforts. LDRD has historically seeded important programs and facilities at the lab. Two of these facilities, the Advanced Photon Source and the Center for Nanoscale Materials, are now vital contributors to today's LDRD Program. New and enhanced capabilities, many of which relied on LDRD in their early stages, now help the laboratory pursue its evolving strategic goals. LDRD has, since its inception, been an invaluable resource for positioning the Laboratory to anticipate, and thus be prepared to contribute to, the future science and technology needs of DOE and the nation. During times of change, LDRD becomes all the more vital for facilitating the necessary adjustments while maintaining and enhancing the capabilities of our staff and facilities. Although I am new to the role of Laboratory Director, my immediate prior service as Deputy Laboratory Director for Programs afforded me continuous involvement in the LDRD program and its management. Therefore, I can attest that Argonne's program adhered closely to the requirements of DOE Order 413.2b and associated guidelines governing LDRD. Our LDRD program management continually strives to be more efficient. In addition

Laboratory Directed Research and Development FY2008 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kammeraad, J E; Jackson, K J; Sketchley, J A

The Laboratory Directed Research and Development (LDRD) Program, authorized by Congress in 1991 and administered by the Institutional Science and Technology Office at Lawrence Livermore, is our primary means for pursuing innovative, long-term, high-risk, and potentially high-payoff research that supports the full spectrum of national security interests encompassed by the missions of the Laboratory, the Department of Energy, and National Nuclear Security Administration. The accomplishments described in this annual report demonstrate the strong alignment of the LDRD portfolio with these missions and contribute to the Laboratory's success in meeting its goals. The LDRD budget of $91.5 million for fiscal yearmore » 2008 sponsored 176 projects. These projects were selected through an extensive peer-review process to ensure the highest scientific quality and mission relevance. Each year, the number of deserving proposals far exceeds the funding available, making the selection a tough one indeed. Our ongoing investments in LDRD have reaped long-term rewards for the Laboratory and the nation. Many Laboratory programs trace their roots to research thrusts that began several years ago under LDRD sponsorship. In addition, many LDRD projects contribute to more than one mission area, leveraging the Laboratory's multidisciplinary team approach to science and technology. Safeguarding the nation from terrorist activity and the proliferation of weapons of mass destruction will be an enduring mission of this Laboratory, for which LDRD will continue to play a vital role. The LDRD Program is a success story. Our projects continue to win national recognition for excellence through prestigious awards, papers published in peer-reviewed journals, and patents granted. With its reputation for sponsoring innovative projects, the LDRD Program is also a major vehicle for attracting and retaining the best and the brightest technical staff and for establishing collaborations with universities

Protein-diazonium adduct direct electrografting onto SPRi-biochip.

PubMed

Corgier, Benjamin P; Bellon, Sophie; Anger-Leroy, Marielle; Blum, Loïc J; Marquette, Christophe A

2009-08-18

A direct protein immobilization method for surface plasmon resonance imaging (SPRi) gold chip arraying is exposed. The biomolecule electroaddressing strategy, previously demonstrated by our team on carbon surfaces, is here valuably involved and adapted to create a straightforward and efficient protein immobilization process onto SPRi-biochips. The proteins, modified with an aryl-diazonium adduct, are addressed to the SPRi chip surface through the electroreduction of the aryl-diazonium. The biomolecule deposition was followed through SPRi live measurements during the electrografting process. A specially designed setup enabled us to directly observe the mass increasing at the sensor surface while the proteins were electrografted. A pin electrospotting method, allowing the achievement of distinct sensing layers on gold SPRi-biochips, was used to generate microarray biochips. The integrity of the immobilized proteins and the specificity of the detection, based on antigen/antibody interactions, were demonstrated for the detection of specific antibodies and ovalbumin. The SPRi detection limit of ovalbumin using the electroaddressing of anti-ovalbumin IgG was compared with two other immobilization procedures, cystamine-glutaraldehyde self-assembled monolayer and pyrrole, and was found to be a decade lower than these ones (100 ng/mL, i.e., 2 nM).

An affinity-directed protein missile system for targeted proteolysis.

PubMed

Fulcher, Luke J; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro; Sapkota, Gopal P

2016-10-01

The von Hippel-Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. © 2016 The Authors.

An affinity-directed protein missile system for targeted proteolysis

PubMed Central

Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

2016-01-01

The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

A 13-week research-based biochemistry laboratory curriculum.

PubMed

Lefurgy, Scott T; Mundorff, Emily C

2017-09-01

Here, we present a 13-week research-based biochemistry laboratory curriculum designed to provide the students with the experience of engaging in original research while introducing foundational biochemistry laboratory techniques. The laboratory experience has been developed around the directed evolution of an enzyme chosen by the instructor, with mutations designed by the students. Ideal enzymes for this curriculum are able to be structurally modeled, solubly expressed, and monitored for activity by UV/Vis spectroscopy, and an example curriculum for haloalkane dehalogenase is given. Unique to this curriculum is a successful implementation of saturation mutagenesis and high-throughput screening of enzyme function, along with bioinformatics analysis, homology modeling, structural analysis, protein expression and purification, polyacrylamide gel electrophoresis, UV/Vis spectroscopy, and enzyme kinetics. Each of these techniques is carried out using a novel student-designed mutant library or enzyme variant unique to the lab team and, importantly, not described previously in the literature. Use of a well-established set of protocols promotes student data quality. Publication may result from the original student-generated hypotheses and data, either from the class as a whole or individual students that continue their independent projects upon course completion. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):437-448, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Selection on Network Dynamics Drives Differential Rates of Protein Domain Evolution

PubMed Central

Mannakee, Brian K.; Gutenkunst, Ryan N.

2016-01-01

The long-held principle that functionally important proteins evolve slowly has recently been challenged by studies in mice and yeast showing that the severity of a protein knockout only weakly predicts that protein’s rate of evolution. However, the relevance of these studies to evolutionary changes within proteins is unknown, because amino acid substitutions, unlike knockouts, often only slightly perturb protein activity. To quantify the phenotypic effect of small biochemical perturbations, we developed an approach to use computational systems biology models to measure the influence of individual reaction rate constants on network dynamics. We show that this dynamical influence is predictive of protein domain evolutionary rate within networks in vertebrates and yeast, even after controlling for expression level and breadth, network topology, and knockout effect. Thus, our results not only demonstrate the importance of protein domain function in determining evolutionary rate, but also the power of systems biology modeling to uncover unanticipated evolutionary forces. PMID:27380265

Evolution of sfbI Encoding Streptococcal Fibronectin-Binding Protein I: Horizontal Genetic Transfer and Gene Mosaic Structure

PubMed Central

Towers, Rebecca J.; Fagan, Peter K.; Talay, Susanne R.; Currie, Bart J.; Sriprakash, Kadaba S.; Walker, Mark J.; Chhatwal, Gursharan S.

2003-01-01

Streptococcal fibronectin-binding protein is an important virulence factor involved in colonization and invasion of epithelial cells and tissues by Streptococcus pyogenes. In order to investigate the mechanisms involved in the evolution of sfbI, the sfbI genes from 54 strains were sequenced. Thirty-four distinct alleles were identified. Three principal mechanisms appear to have been involved in the evolution of sfbI. The amino-terminal aromatic amino acid-rich domain is the most variable region and is apparently generated by intergenic recombination of horizontally acquired DNA cassettes, resulting in a genetic mosaic in this region. Two distinct and divergent sequence types that shared only 61 to 70% identity were identified in the central proline-rich region, while variation at the 3′ end of the gene is due to deletion or duplication of defined repeat units. Potential antigenic and functional variabilities in SfbI imply significant selective pressure in vivo with direct implications for the microbial pathogenesis of S. pyogenes. PMID:14662917

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution.

PubMed

Ye, Lidan; Yang, Chengcheng; Yu, Hongwei

2018-01-01

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature's biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell

PubMed Central

Holthaus, Karin Brigit; Strasser, Bettina; Sipos, Wolfgang; Schmidt, Heiko A.; Mlitz, Veronika; Sukseree, Supawadee; Weissenbacher, Anton; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

2016-01-01

The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation. PMID:26601937

Sexual selection and the adaptive evolution of PKDREJ protein in primates and rodents

PubMed Central

Vicens, Alberto; Gómez Montoto, Laura; Couso-Ferrer, Francisco; Sutton, Keith A.; Roldan, Eduardo R.S.

2015-01-01

PKDREJ is a testis-specific protein thought to be located on the sperm surface. Functional studies in the mouse revealed that loss of PKDREJ has effects on sperm transport and the ability to undergo an induced acrosome reaction. Thus, PKDREJ has been considered a potential target of post-copulatory sexual selection in the form of sperm competition. Proteins involved in reproductive processes often show accelerated evolution. In many cases, this rapid divergence is promoted by positive selection which may be driven, at least in part, by post-copulatory sexual selection. We analysed the evolution of the PKDREJ protein in primates and rodents and assessed whether PKDREJ divergence is associated with testes mass relative to body mass, which is a reliable proxy of sperm competition levels. Evidence of an association between the evolutionary rate of the PKDREJ gene and testes mass relative to body mass was not found in primates. Among rodents, evidence of positive selection was detected in the Pkdrej gene in the family Cricetidae but not in Muridae. We then assessed whether Pkdrej divergence is associated with episodes of sperm competition in these families. We detected a positive significant correlation between the evolutionary rates of Pkdrej and testes mass relative to body mass in cricetids. These findings constitute the first evidence of post-copulatory sexual selection influencing the evolution of a protein that participates in the mechanisms regulating sperm transport and the acrosome reaction, strongly suggesting that positive selection may act on these fertilization steps, leading to advantages in situations of sperm competition. PMID:25304980

Laboratory directed research and development FY98 annual report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Ayat, R; Holzrichter, J

1999-05-01

In 1984, Congress and the Department of Energy (DOE) established the Laboratory Directed Research and Development (LDRD) Program to enable the director of a national laboratory to foster and expedite innovative research and development (R and D) in mission areas. The Lawrence Livermore National Laboratory (LLNL) continually examines these mission areas through strategic planning and shapes the LDRD Program to meet its long-term vision. The goal of the LDRD Program is to spur development of new scientific and technical capabilities that enable LLNL to respond to the challenges within its evolving mission areas. In addition, the LDRD Program provides LLNLmore » with the flexibility to nurture and enrich essential scientific and technical competencies and enables the Laboratory to attract the most qualified scientists and engineers. The FY98 LDRD portfolio described in this annual report has been carefully structured to continue the tradition of vigorously supporting DOE and LLNL strategic vision and evolving mission areas. The projects selected for LDRD funding undergo stringent review and selection processes, which emphasize strategic relevance and require technical peer reviews of proposals by external and internal experts. These FY98 projects emphasize the Laboratory's national security needs: stewardship of the U.S. nuclear weapons stockpile, responsibility for the counter- and nonproliferation of weapons of mass destruction, development of high-performance computing, and support of DOE environmental research and waste management programs.« less

Sexy sons from re-mating do not recoup the direct costs of harmful male interactions in the Drosophila melanogaster laboratory model system.

PubMed

Orteiza, N; Linder, J E; Rice, W R

2005-09-01

The empirical foundation for sexual conflict theory is the data from many different taxa demonstrating that females are harmed while interacting with males. However, the interpretation of this keystone evidence has been challenged because females may more than counterbalance the direct costs of interacting with males by the indirect benefits of obtaining higher quality genes for their offspring. A quantification of this trade-off is critical to resolve the controversy and is presented here. A multi-generation fitness assay in the Drosophila melanogaster laboratory model system was used to quantify both the direct costs to females due to interactions with males and indirect benefits via sexy sons. We specifically focus on the interactions that occur between males and nonvirgin females. In the laboratory environment of our base population, females mate soon after eclosion and store sufficient sperm for their entire lifetime, yet males persistently court these nonvirgin females and frequently succeed in re-mating them. Females may benefit from these interactions despite direct costs to their lifetime fecundity if re-mating allows them to trade-up to mates of higher genetic quality and thereby secure indirect benefits for their offspring. We found that direct costs of interactions between males and nonvirgin females substantially exceeded indirect benefits through sexy sons. These data, in combination with past studies of the good genes route of indirect benefits, demonstrate that inter-sexual interactions drive sexually antagonistic co-evolution in this model system.

Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins

NASA Technical Reports Server (NTRS)

Kretsinger, R. H.; Nakayama, S.

1993-01-01

In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

Evolution of the acyl-CoA binding protein (ACBP)

PubMed Central

Burton, Mark; Rose, Timothy M.; Færgeman, Nils J.; Knudsen, Jens

2005-01-01

Acyl-CoA-binding protein (ACBP) is a 10 kDa protein that binds C12–C22 acyl-CoA esters with high affinity. In vitro and in vivo experiments suggest that it is involved in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, regulation of the intracellular acyl-CoA pool size, donation of acyl-CoA esters for β-oxidation, vesicular trafficking, complex lipid synthesis and gene regulation. In the present study, we delineate the evolutionary history of ACBP to get a complete picture of its evolution and distribution among species. ACBP homologues were identified in all four eukaryotic kingdoms, Animalia, Plantae, Fungi and Protista, and eleven eubacterial species. ACBP homologues were not detected in any other known bacterial species, or in archaea. Nearly all of the ACBP-containing bacteria are pathogenic to plants or animals, suggesting that an ACBP gene could have been acquired from a eukaryotic host by horizontal gene transfer. Many bacterial, fungal and higher eukaryotic species only harbour a single ACBP homologue. However, a number of species, ranging from protozoa to vertebrates, have evolved two to six lineage-specific paralogues through gene duplication and/or retrotransposition events. The ACBP protein is highly conserved across phylums, and the majority of ACBP genes are subjected to strong purifying selection. Experimental evidence indicates that the function of ACBP has been conserved from yeast to humans and that the multiple lineage-specific paralogues have evolved altered functions. The appearance of ACBP very early on in evolution points towards a fundamental role of ACBP in acyl-CoA metabolism, including ceramide synthesis and in signalling. PMID:16018771

The TALE face of Hox proteins in animal evolution

PubMed Central

Merabet, Samir; Galliot, Brigitte

2015-01-01

Hox genes are major regulators of embryonic development. One of their most conserved functions is to coordinate the formation of specific body structures along the anterior-posterior (AP) axis in Bilateria. This architectural role was at the basis of several morphological innovations across bilaterian evolution. In this review, we traced the origin of the Hox patterning system by considering the partnership with PBC and Meis proteins. PBC and Meis belong to the TALE-class of homeodomain-containing transcription factors and act as generic cofactors of Hox proteins for AP axis patterning in Bilateria. Recent data indicate that Hox proteins acquired the ability to interact with their TALE partners in the last common ancestor of Bilateria and Cnidaria. These interactions relied initially on a short peptide motif called hexapeptide (HX), which is present in Hox and non-Hox protein families. Remarkably, Hox proteins can also recruit the TALE cofactors by using specific PBC Interaction Motifs (SPIMs). We describe how a functional Hox/TALE patterning system emerged in eumetazoans through the acquisition of SPIMs. We anticipate that interaction flexibility could be found in other patterning systems, being at the heart of the astonishing morphological diversity observed in the animal kingdom. PMID:26347770

A PDB-wide, evolution-based assessment of protein-protein interfaces.

PubMed

Baskaran, Kumaran; Duarte, Jose M; Biyani, Nikhil; Bliven, Spencer; Capitani, Guido

2014-10-18

Thanks to the growth in sequence and structure databases, more than 50 million sequences are now available in UniProt and 100,000 structures in the PDB. Rich information about protein-protein interfaces can be obtained by a comprehensive study of protein contacts in the PDB, their sequence conservation and geometric features. An automated computational pipeline was developed to run our Evolutionary Protein-Protein Interface Classifier (EPPIC) software on the entire PDB and store the results in a relational database, currently containing > 800,000 interfaces. This allows the analysis of interface data on a PDB-wide scale. Two large benchmark datasets of biological interfaces and crystal contacts, each containing about 3000 entries, were automatically generated based on criteria thought to be strong indicators of interface type. The BioMany set of biological interfaces includes NMR dimers solved as crystal structures and interfaces that are preserved across diverse crystal forms, as catalogued by the Protein Common Interface Database (ProtCID) from Xu and Dunbrack. The second dataset, XtalMany, is derived from interfaces that would lead to infinite assemblies and are therefore crystal contacts. BioMany and XtalMany were used to benchmark the EPPIC approach. The performance of EPPIC was also compared to classifications from the Protein Interfaces, Surfaces, and Assemblies (PISA) program on a PDB-wide scale, finding that the two approaches give the same call in about 88% of PDB interfaces. By comparing our safest predictions to the PDB author annotations, we provide a lower-bound estimate of the error rate of biological unit annotations in the PDB. Additionally, we developed a PyMOL plugin for direct download and easy visualization of EPPIC interfaces for any PDB entry. Both the datasets and the PyMOL plugin are available at http://www.eppic-web.org/ewui/\\#downloads. Our computational pipeline allows us to analyze protein-protein contacts and their sequence

Linear motif-mediated interactions have contributed to the evolution of modularity in complex protein interaction networks.

PubMed

Kim, Inhae; Lee, Heetak; Han, Seong Kyu; Kim, Sanguk

2014-10-01

The modular architecture of protein-protein interaction (PPI) networks is evident in diverse species with a wide range of complexity. However, the molecular components that lead to the evolution of modularity in PPI networks have not been clearly identified. Here, we show that weak domain-linear motif interactions (DLIs) are more likely to connect different biological modules than strong domain-domain interactions (DDIs). This molecular division of labor is essential for the evolution of modularity in the complex PPI networks of diverse eukaryotic species. In particular, DLIs may compensate for the reduction in module boundaries that originate from increased connections between different modules in complex PPI networks. In addition, we show that the identification of biological modules can be greatly improved by including molecular characteristics of protein interactions. Our findings suggest that transient interactions have played a unique role in shaping the architecture and modularity of biological networks over the course of evolution.

Argonne National Laboratory Annual Report of Laboratory Directed Research and Development program activities FY 2011.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Office of The Director)

As a national laboratory Argonne concentrates on scientific and technological challenges that can only be addressed through a sustained, interdisciplinary focus at a national scale. Argonne's eight major initiatives, as enumerated in its strategic plan, are Hard X-ray Sciences, Leadership Computing, Materials and Molecular Design and Discovery, Energy Storage, Alternative Energy and Efficiency, Nuclear Energy, Biological and Environmental Systems, and National Security. The purposes of Argonne's Laboratory Directed Research and Development (LDRD) Program are to encourage the development of novel technical concepts, enhance the Laboratory's research and development (R and D) capabilities, and pursue its strategic goals. projects are selectedmore » from proposals for creative and innovative R and D studies that require advance exploration before they are considered to be sufficiently developed to obtain support through normal programmatic channels. Among the aims of the projects supported by the LDRD Program are the following: establishment of engineering proof of principle, assessment of design feasibility for prospective facilities, development of instrumentation or computational methods or systems, and discoveries in fundamental science and exploratory development.« less

Conservation of protein structure over four billion years.

PubMed

Ingles-Prieto, Alvaro; Ibarra-Molero, Beatriz; Delgado-Delgado, Asuncion; Perez-Jimenez, Raul; Fernandez, Julio M; Gaucher, Eric A; Sanchez-Ruiz, Jose M; Gavira, Jose A

2013-09-03

Little is known about the evolution of protein structures and the degree of protein structure conservation over planetary time scales. Here, we report the X-ray crystal structures of seven laboratory resurrections of Precambrian thioredoxins dating up to approximately four billion years ago. Despite considerable sequence differences compared with extant enzymes, the ancestral proteins display the canonical thioredoxin fold, whereas only small structural changes have occurred over four billion years. This remarkable degree of structure conservation since a time near the last common ancestor of life supports a punctuated-equilibrium model of structure evolution in which the generation of new folds occurs over comparatively short periods and is followed by long periods of structural stasis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Microbial Evolution at High Pressure: Deep Sea and Laboratory Studies

NASA Astrophysics Data System (ADS)

Bartlett, D. H.

2011-12-01

Elevated hydrostatic pressures are present in deep-sea and deep-Earth environments where this physical parameter has influenced the evolution and characteristics of life. Piezophilic (high-pressure-adapted) microbes have been isolated from diverse deep-sea settings, and would appear likely to occur in deep-subsurface habitats as well. In order to discern the factors enabling life at high pressure my research group has explored these adaptations at various levels, most recently including molecular analyses of deep-sea trench communities, and through the selective evolution of the model microbe Escherichia coli in the laboratory to progressively higher pressures. Much of the field work has focused on the microbes present in the deeper portions of the Puerto Rico Trench (PRT)and in the Peru-Chile Trench (PCT), from 6-8.5 km below the sea surface (~60-85 megapascals pressure). Culture-independent phylogenetic data on the Bacteria and Archaea present on particles or free-living, along with data on the microeukarya present was complemented with genomic analyses and the isolation and characterization of microbes in culture. Metagenomic analyses of the PRT revealed increased genome sizes and an overrepresentation at depth of sulfatases for the breakdown of sulfated polysaccharides and specific categories of transporters, including those associated with the transport of diverse cations or carboxylate ions, or associated with heavy metal resistance. Single-cell genomic studies revealed several linneages which recruited to the PRT metagenome far better than existing marine microbial genome sequences. analyses. Novel high pressure culture approaches have yielded new piezophiles including species preferring very low nutrient levels, those living off of hydrocarbons, and those adapted to various electron donor/electron acceptor combinations. In order to more specifically focus on functions enabling life at increased pressure selective evolution experiments were performed with

Evolution of a residue laboratory network and the management tools for monitoring its performance.

PubMed

Lins, E S; Conceição, E S; Mauricio, A De Q

2012-01-01

Since 2005 the National Residue & Contaminants Control Plan (NRCCP) in Brazil has been considerably enhanced, increasing the number of samples, substances and species monitored, and also the analytical detection capability. The Brazilian laboratory network was forced to improve its quality standards in order to comply with the NRCP's own evolution. Many aspects such as the limits of quantification (LOQs), the quality management systems within the laboratories and appropriate method validation are in continuous improvement, generating new scenarios and demands. Thus, efficient management mechanisms for monitoring network performance and its adherence to the established goals and guidelines are required. Performance indicators associated to computerised information systems arise as a powerful tool to monitor the laboratories' activity, making use of different parameters to describe this activity on a day-to-day basis. One of these parameters is related to turnaround times, and this factor is highly affected by the way each laboratory organises its management system, as well as the regulatory requirements. In this paper a global view is presented of the turnaround times related to the type of analysis, laboratory, number of samples per year, type of matrix, country region and period of the year, all these data being collected from a computerised system called SISRES. This information gives a solid background to management measures aiming at the improvement of the service offered by the laboratory network.

Comparative analysis of protein evolution in the genome of pre-epidemic and epidemic Zika virus.

PubMed

Ramaiah, Arunachalam; Dai, Lei; Contreras, Deisy; Sinha, Sanjeev; Sun, Ren; Arumugaswami, Vaithilingaraja

2017-07-01

Zika virus (ZIKV) causes microcephaly in congenital infection, neurological disorders, and poor pregnancy outcome and no vaccine is available for use in humans or approved. Although ZIKV was first discovered in 1947, the exact mechanism of virus replication and pathogenesis remains unknown. Recent outbreaks of Zika virus in the Americas clearly suggest a human-mosquito cycle or urban cycle of transmission. Understanding the conserved and adaptive features in the evolution of ZIKV genome will provide a hint on the mechanism of ZIKV adaptation to a new cycle of transmission. Here, we show comprehensive analysis of protein evolution of ZIKV strains including the current 2015-16 outbreak. To identify the constraints on ZIKV evolution, selection pressure at individual codons, immune epitopes and co-evolving sites were analyzed. Phylogenetic trees show that the ZIKV strains of the Asian genotype form distinct cluster and share a common ancestor with African genotype. The TMRCA (Time to the Most Recent Common Ancestor) for the Asian lineage and the subsequently evolved Asian human strains was calculated at 88 and 34years ago, respectively. The proteome of current 2015/16 epidemic ZIKV strains of Asian genotype was found to be genetically conserved due to genome-wide negative selection, with limited positive selection. We identified a total of 16 amino acid substitutions in the epidemic and pre-epidemic strains from human, mosquito, and monkey hosts. Negatively selected amino acid sites of Envelope protein (E-protein) (positions 69, 166, and 174) and NS5 (292, 345, and 587) were located in central dimerization domains and C-terminal RNA-directed RNA polymerase regions, respectively. The predicted 137 (92 CD4 TCEs; 45 CD8 TCEs) immunogenic peptide chains comprising negatively selected amino acid sites can be considered as suitable target for sub-unit vaccine development, as these sites are less likely to generate immune-escape variants due to strong functional constrains

[Laboratory diagnosis of genital herpes--direct immunofluorescence method].

PubMed

Majewska, Anna; Romejko-Wolniewicz, Ewa; Zareba-Szczudlik, Julia; Kilijańczyk, Marek; Gajewska, Małgorzata; Młynarczyk, Grazyna

2013-07-01

Aim of the study was to determine clinical usefulness of direct immunofluorescence method in the laboratory diagnosis of genital herpes in women. Overall 187 anogenital swabs were collected from 120 women. Using a dacron-tipped applicator 83 swabs were collected from women suspected of genital herpes and 104 from patients with no signs of genital infection. All samples were tested using cell culture (Vero cell line) and then direct immunofluorescence method (DIF) for the identification of antigens of herpes simplex viruses: HSV-1 and HSV-2. Characteristic cytopathic effect (CPE), indicative of alphaherpesvirus infection, was observed in 43.4% of cultures with clinical specimens collected from women with suspected genital herpes and in 29.8% of cultures of clinical specimens taken from patients with no clinical symptoms of genital herpes. Herpes simplex viruses were determined in 73 samples by direct immunofluorescence method after amplification of the virus in cell culture. The DIF test confirmed the diagnosis based on the microscopic CPE observation in 85%. In 15% of samples (taken from pregnant women without clinical signs of infection) we reported positive immunofluorescence in the absence of CPE. The frequency of antigen detection was statistically significantly higher in samples that were positive by culture study (chi-square test with Yates's correction, p < 0.01). This method proved to be highly sensitive (97%) in women with clinically suspected infection. High negative predictive value (99%) proves the clinical utility of the DIF in these group of patients. In asymptomatic infections, viral antigens were detected most frequently in the swabs from the cervical canal, and in cases of suspected genital herpes in swabs taken from the vestibule of the vagina and the vulva. However, there was no statistically significant difference in the frequency of detection of Herpes Simplex Virus antigens in specimens from different parts of the genital tract in both groups

Rapid Modeling and Analysis Tools: Evolution, Status, Needs and Directions

NASA Technical Reports Server (NTRS)

Knight, Norman F., Jr.; Stone, Thomas J.; Ransom, Jonathan B. (Technical Monitor)

2002-01-01

Advanced aerospace systems are becoming increasingly more complex, and customers are demanding lower cost, higher performance, and high reliability. Increased demands are placed on the design engineers to collaborate and integrate design needs and objectives early in the design process to minimize risks that may occur later in the design development stage. High performance systems require better understanding of system sensitivities much earlier in the design process to meet these goals. The knowledge, skills, intuition, and experience of an individual design engineer will need to be extended significantly for the next generation of aerospace system designs. Then a collaborative effort involving the designer, rapid and reliable analysis tools and virtual experts will result in advanced aerospace systems that are safe, reliable, and efficient. This paper discusses the evolution, status, needs and directions for rapid modeling and analysis tools for structural analysis. First, the evolution of computerized design and analysis tools is briefly described. Next, the status of representative design and analysis tools is described along with a brief statement on their functionality. Then technology advancements to achieve rapid modeling and analysis are identified. Finally, potential future directions including possible prototype configurations are proposed.

1996 Laboratory directed research and development annual report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyers, C.E.; Harvey, C.L.; Lopez-Andreas, L.M.

This report summarizes progress from the Laboratory Directed Research and Development (LDRD) program during fiscal year 1996. In addition to a programmatic and financial overview, the report includes progress reports from 259 individual R&D projects in seventeen categories. The general areas of research include: engineered processes and materials; computational and information sciences; microelectronics and photonics; engineering sciences; pulsed power; advanced manufacturing technologies; biomedical engineering; energy and environmental science and technology; advanced information technologies; counterproliferation; advanced transportation; national security technology; electronics technologies; idea exploration and exploitation; production; and science at the interfaces - engineering with atoms.

Loss of the insulator protein CTCF during nematode evolution

PubMed Central

Heger, Peter; Marin, Birger; Schierenberg, Einhard

2009-01-01

Background The zinc finger (ZF) protein CTCF (CCCTC-binding factor) is highly conserved in Drosophila and vertebrates where it has been shown to mediate chromatin insulation at a genomewide level. A mode of genetic regulation that involves insulators and insulator binding proteins to establish independent transcriptional units is currently not known in nematodes including Caenorhabditis elegans. We therefore searched in nematodes for orthologs of proteins that are involved in chromatin insulation. Results While orthologs for other insulator proteins were absent in all 35 analysed nematode species, we find orthologs of CTCF in a subset of nematodes. As an example for these we cloned the Trichinella spiralis CTCF-like gene and revealed a genomic structure very similar to the Drosophila counterpart. To investigate the pattern of CTCF occurrence in nematodes, we performed phylogenetic analysis with the ZF protein sets of completely sequenced nematodes. We show that three ZF proteins from three basal nematodes cluster together with known CTCF proteins whereas no zinc finger protein of C. elegans and other derived nematodes does so. Conclusion Our findings show that CTCF and possibly chromatin insulation are present in basal nematodes. We suggest that the insulator protein CTCF has been secondarily lost in derived nematodes like C. elegans. We propose a switch in the regulation of gene expression during nematode evolution, from the common vertebrate and insect type involving distantly acting regulatory elements and chromatin insulation to a so far poorly characterised mode present in more derived nematodes. Here, all or some of these components are missing. Instead operons, polycistronic transcriptional units common in derived nematodes, seemingly adopted their function. PMID:19712444

Blood tolerant laccase by directed evolution.

PubMed

Mate, Diana M; Gonzalez-Perez, David; Falk, Magnus; Kittl, Roman; Pita, Marcos; De Lacey, Antonio L; Ludwig, Roland; Shleev, Sergey; Alcalde, Miguel

2013-02-21

High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnology. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific composition of human blood (above neutral pH, high chloride concentration) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chemical syntheses, and detoxification. Copyright © 2013 Elsevier Ltd. All rights reserved.

Laboratory Directed Research and Development Program FY 2008 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

editor, Todd C Hansen

2009-02-23

The Ernest Orlando Lawrence Berkeley National Laboratory (Berkeley Lab or LBNL) is a multi-program national research facility operated by the University of California for the Department of Energy (DOE). As an integral element of DOE's National Laboratory System, Berkeley Lab supports DOE's missions in fundamental science, energy resources, and environmental quality. Berkeley Lab programs advance four distinct goals for DOE and the nation: (1) To perform leading multidisciplinary research in the computing sciences, physical sciences, energy sciences, biosciences, and general sciences in a manner that ensures employee and public safety and protection of the environment. (2) To develop and operatemore » unique national experimental facilities for qualified investigators. (3) To educate and train future generations of scientists and engineers to promote national science and education goals. (4) To transfer knowledge and technological innovations and to foster productive relationships among Berkeley Lab's research programs, universities, and industry in order to promote national economic competitiveness. Berkeley Lab's research and the Laboratory Directed Research and Development (LDRD) program support DOE's Strategic Themes that are codified in DOE's 2006 Strategic Plan (DOE/CF-0010), with a primary focus on Scientific Discovery and Innovation. For that strategic theme, the Fiscal Year (FY) 2008 LDRD projects support each one of the three goals through multiple strategies described in the plan. In addition, LDRD efforts support the four goals of Energy Security, the two goals of Environmental Responsibility, and Nuclear Security (unclassified fundamental research that supports stockpile safety and nonproliferation programs). The LDRD program supports Office of Science strategic plans, including the 20-year Scientific Facilities Plan and the Office of Science Strategic Plan. The research also supports the strategic directions periodically under consideration and

Protein crystal growth results from the United States Microgravity Laboratory-1 mission

NASA Technical Reports Server (NTRS)

Delucas, Lawrence J.; Moore, K. M.; Vanderwoerd, M.; Bray, T. L.; Smith, C.; Carson, M.; Narayana, S. V. L.; Rosenblum, W. M.; Carter, D.; Clark, A. D, Jr.

1994-01-01

Protein crystal growth experiments have been performed by this laboratory on 18 Space Shuttle missions since April, 1985. In addition, a number of microgravity experiments also have been performed and reported by other investigators. These Space Shuttle missions have been used to grow crystals of a variety of proteins using vapor diffusion, liquid diffusion, and temperature-induced crystallization techniques. The United States Microgravity Laboratory - 1 mission (USML-1, June 25 - July 9, 1992) was a Spacelab mission dedicated to experiments involved in materials processing. New protein crystal growth hardware was developed to allow in orbit examination of initial crystal growth results, the knowledge from which was used on subsequent days to prepare new crystal growth experiments. In addition, new seeding hardware and techniques were tested as well as techniques that would prepare crystals for analysis by x-ray diffraction, a capability projected for the planned Space Station. Hardware that was specifically developed for the USML-1 mission will be discussed along with the experimental results from this mission.

LDRD 2014 Annual Report: Laboratory Directed Research and Development Program Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatton, Diane

Each year, Brookhaven National Laboratory (BNL) is required to provide a program description and overview of its Laboratory Directed Research and Development Program (LDRD) to the Department of Energy (DOE) in accordance with DOE Order 413.2B dated April 19, 2006. This report provides a detailed look at the scientific and technical activities for each of the LDRD projects funded by BNL in FY 2014, as required. In FY 2014, the BNL LDRD Program funded 40 projects, 8 of which were new starts, at a total cost of $9.6M.

LDRD 2012 Annual Report: Laboratory Directed Research and Development Program Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bookless, William

Each year, Brookhaven National Laboratory (BNL) is required to provide a program description and overview of its Laboratory Directed Research and Development Program (LDRD) to the Department of Energy in accordance with DOE Order 413.2B dated April 19, 2006. This report provides a detailed look at the scientific and technical activities for each of the LDRD projects funded by BNL in FY2012, as required. In FY2012, the BNL LDRD Program funded 52 projects, 14 of which were new starts, at a total cost of $10,061,292.

LDRD 2015 Annual Report: Laboratory Directed Research and Development Program Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatton, D.

Each year, Brookhaven National Laboratory (BNL) is required to provide a program description and overview of its Laboratory Directed Research and Development Program (LDRD) to the Department of Energy (DOE) in accordance with DOE Order 413.2B dated April 19, 2006. This report provides a detailed look at the scientific and technical activities for each of the LDRD projects funded by BNL in FY 2015, as required. In FY 2015, the BNL LDRD Program funded 43 projects, 12 of which were new starts, at a total cost of $9.5M.

In silico modelling of directed evolution: Implications for experimental design and stepwise evolution.

PubMed

Wedge, David C; Rowe, William; Kell, Douglas B; Knowles, Joshua

2009-03-07

We model the process of directed evolution (DE) in silico using genetic algorithms. Making use of the NK fitness landscape model, we analyse the effects of mutation rate, crossover and selection pressure on the performance of DE. A range of values of K, the epistatic interaction of the landscape, are considered, and high- and low-throughput modes of evolution are compared. Our findings suggest that for runs of or around ten generations' duration-as is typical in DE-there is little difference between the way in which DE needs to be configured in the high- and low-throughput regimes, nor across different degrees of landscape epistasis. In all cases, a high selection pressure (but not an extreme one) combined with a moderately high mutation rate works best, while crossover provides some benefit but only on the less rugged landscapes. These genetic algorithms were also compared with a "model-based approach" from the literature, which uses sequential fixing of the problem parameters based on fitting a linear model. Overall, we find that purely evolutionary techniques fare better than do model-based approaches across all but the smoothest landscapes.

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

PubMed

Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2015-10-20

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

Recent progress and future directions in protein-protein docking.

PubMed

Ritchie, David W

2008-02-01

This article gives an overview of recent progress in protein-protein docking and it identifies several directions for future research. Recent results from the CAPRI blind docking experiments show that docking algorithms are steadily improving in both reliability and accuracy. Current docking algorithms employ a range of efficient search and scoring strategies, including e.g. fast Fourier transform correlations, geometric hashing, and Monte Carlo techniques. These approaches can often produce a relatively small list of up to a few thousand orientations, amongst which a near-native binding mode is often observed. However, despite the use of improved scoring functions which typically include models of desolvation, hydrophobicity, and electrostatics, current algorithms still have difficulty in identifying the correct solution from the list of false positives, or decoys. Nonetheless, significant progress is being made through better use of bioinformatics, biochemical, and biophysical information such as e.g. sequence conservation analysis, protein interaction databases, alanine scanning, and NMR residual dipolar coupling restraints to help identify key binding residues. Promising new approaches to incorporate models of protein flexibility during docking are being developed, including the use of molecular dynamics snapshots, rotameric and off-rotamer searches, internal coordinate mechanics, and principal component analysis based techniques. Some investigators now use explicit solvent models in their docking protocols. Many of these approaches can be computationally intensive, although new silicon chip technologies such as programmable graphics processor units are beginning to offer competitive alternatives to conventional high performance computer systems. As cryo-EM techniques improve apace, docking NMR and X-ray protein structures into low resolution EM density maps is helping to bridge the resolution gap between these complementary techniques. The use of symmetry and

Microstructural Evolution in Intensively Melt Sheared Direct Chill Cast Al-Alloys

NASA Astrophysics Data System (ADS)

Jones, S.; Rao, A. K. Prasada; Patel, J. B.; Scamans, G. M.; Fan, Z.

The work presented here introduces the novel melt conditioned direct chill casting (MC-DC) technology, where intensive melt shearing is applied to the conventional direct-chill casting process. MC-DC casting can successfully produce high quality Al-alloy billets. The results obtained from 80 mm diameter billets cast at speed of 200 mm/min show that MC-DC casting of Al-alloys, substantially refines the microstructure and reduces macro-segregation. In this paper, we present the preliminary results and discuss microstructural evolution during MC-DC casting of Al-alloys.

Ancestral gene reconstruction and synthesis of ancient rhodopsins in the laboratory.

PubMed

Chang, Belinda S W

2003-08-01

Laboratory synthesis of ancestral proteins offers an intriguing opportunity to study the past directly. The development of Bayesian methods to infer ancestral sequences, combined with advances in models of molecular evolution, and synthetic gene technology make this an increasingly promising approach in evolutionary studies of molecular function. Visual pigments form the first step in the biochemical cascade of events in the retina in all animals known to possess visual capabilities. In vertebrates, the necessity of spanning a dynamic range of light intensities of many orders of magnitude has given rise to two different types of photoreceptors, rods specialized for dim-light conditions, and cones for daylight and color vision. These photoreceptors contain different types of visual pigment genes. Reviewed here are methods of inferring ancestral sequences, chemical synthesis of artificial ancestral genes in the laboratory, and applications to the evolution of vertebrate visual systems and the experimental recreation of an archosaur rod visual pigment. The ancestral archosaurs gave rise to several notable lineages of diapsid reptiles, including the birds and the dinosaurs, and would have existed over 200 MYA. What little is known of their physiology comes from fossil remains, and inference based on the biology of their living descendants. Despite its age, an ancestral archosaur pigment was successfully recreated in the lab, and showed interesting properties of its wavelength sensitivity that may have implications for the visual capabilities of the ancestral archosaurs in dim light.

Directed evolution of an endoinulinase from Talaromyces purpureogenus toward efficient production of inulooligosaccharides.

PubMed

Afriat-Jurnou, Livnat; Cohen, Rami; Paluy, Irina; Ben-Adiva, Ran; Yadid, Itamar

2018-02-01

Inulinases are fructofuranosyl hydrolases that target the β-2,1 linkage of inulin and hydrolyze it into fructose, glucose and inulooligosaccharides (IOS), the latter are of growing interest as dietary fibers. Inulinases from various microorganisms have been purified, characterized and produced for industrial applications. However, there remains a need for inulinases with increased catalytic activity and better production yields to improve the hydrolysis process and fulfill the growing industrial demands for specific fibers. In this study, we used directed enzyme evolution to increase the yield and activity of an endoinulinase enzyme originated from the filamentous fungus Talaromyces purpureogenus (Penicillium purpureogenum ATCC4713). Our directed evolution approach yielded variants showing up to fivefold improvements in soluble enzyme production compared to the starting point which enabled high-yield production of highly purified recombinant enzyme. The distribution of the enzymatic reaction products demonstrated that after 24 h of incubation, the main product (57%) had a degree of polymerization of 3 (DP3). To the best of our knowledge, this is the first application of directed enzyme evolution to improve inulooligosaccharide production. The approach enabled the screening of large genetic libraries within short time frames and facilitated screening for improved enzymatic activities and properties, such as substrate specificity, product range, thermostability and pH optimum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Studying the co-evolution of protein families with the Mirrortree web server.

PubMed

Ochoa, David; Pazos, Florencio

2010-05-15

The Mirrortree server allows to graphically and interactively study the co-evolution of two protein families, and investigate their possible interactions and functional relationships in a taxonomic context. The server includes the possibility of starting from single sequences and hence it can be used by non-expert users. The web server is freely available at http://csbg.cnb.csic.es/mtserver. It was tested in the main web browsers. Adobe Flash Player is required at the client side to perform the interactive assessment of co-evolution. pazos@cnb.csic.es Supplementary data are available at Bioinformatics online.

Teaching Protein Purification and Characterization Techniques: A Student-Initiated, Project-Oriented Biochemistry Laboratory Course

ERIC Educational Resources Information Center

MacDonald, Gina

2008-01-01

This report describes a biochemistry laboratory that is completely project-oriented. Upper-level biology and chemistry majors work in teams to purify a protein of their choice. After the student groups have completed literature searches, ordered reagents, and made buffers they continue to learn basic protein purification and biochemical techniques…

Two-step relaxation mode analysis with multiple evolution times applied to all-atom molecular dynamics protein simulation.

PubMed

Karasawa, N; Mitsutake, A; Takano, H

2017-12-01

Proteins implement their functionalities when folded into specific three-dimensional structures, and their functions are related to the protein structures and dynamics. Previously, we applied a relaxation mode analysis (RMA) method to protein systems; this method approximately estimates the slow relaxation modes and times via simulation and enables investigation of the dynamic properties underlying the protein structural fluctuations. Recently, two-step RMA with multiple evolution times has been proposed and applied to a slightly complex homopolymer system, i.e., a single [n]polycatenane. This method can be applied to more complex heteropolymer systems, i.e., protein systems, to estimate the relaxation modes and times more accurately. In two-step RMA, we first perform RMA and obtain rough estimates of the relaxation modes and times. Then, we apply RMA with multiple evolution times to a small number of the slowest relaxation modes obtained in the previous calculation. Herein, we apply this method to the results of principal component analysis (PCA). First, PCA is applied to a 2-μs molecular dynamics simulation of hen egg-white lysozyme in aqueous solution. Then, the two-step RMA method with multiple evolution times is applied to the obtained principal components. The slow relaxation modes and corresponding relaxation times for the principal components are much improved by the second RMA.

Two-step relaxation mode analysis with multiple evolution times applied to all-atom molecular dynamics protein simulation

NASA Astrophysics Data System (ADS)

Karasawa, N.; Mitsutake, A.; Takano, H.

2017-12-01

Proteins implement their functionalities when folded into specific three-dimensional structures, and their functions are related to the protein structures and dynamics. Previously, we applied a relaxation mode analysis (RMA) method to protein systems; this method approximately estimates the slow relaxation modes and times via simulation and enables investigation of the dynamic properties underlying the protein structural fluctuations. Recently, two-step RMA with multiple evolution times has been proposed and applied to a slightly complex homopolymer system, i.e., a single [n ] polycatenane. This method can be applied to more complex heteropolymer systems, i.e., protein systems, to estimate the relaxation modes and times more accurately. In two-step RMA, we first perform RMA and obtain rough estimates of the relaxation modes and times. Then, we apply RMA with multiple evolution times to a small number of the slowest relaxation modes obtained in the previous calculation. Herein, we apply this method to the results of principal component analysis (PCA). First, PCA is applied to a 2-μ s molecular dynamics simulation of hen egg-white lysozyme in aqueous solution. Then, the two-step RMA method with multiple evolution times is applied to the obtained principal components. The slow relaxation modes and corresponding relaxation times for the principal components are much improved by the second RMA.

Laboratory Directed Research and Development Program FY2011

DOE Office of Scientific and Technical Information (OSTI.GOV)

none, none

2012-04-27

Berkeley Lab's research and the Laboratory Directed Research and Development (LDRD) program support DOE's Strategic Themes that are codified in DOE's 2006 Strategic Plan (DOE/CF-0010), with a primary focus on Scientific Discovery and Innovation. For that strategic theme, the Fiscal Year (FY) 2011 LDRD projects support each one of the three goals through multiple strategies described in the plan. In addition, LDRD efforts support the four goals of Energy Security, the two goals of Environmental Responsibility, and Nuclear Security (unclassified fundamental research that supports stockpile safety and nonproliferation programs). Going forward in FY 2012, the LDRD program also supports themore » Goals codified in the new DOE Strategic Plan of May, 2011. The LDRD program also supports Office of Science strategic plans, including the 20-year Scientific Facilities Plan and the Office of Science Strategic Plan. The research also supports the strategic directions periodically under consideration and review by the Office of Science Program Offices, such as LDRD projects germane to new research facility concepts and new fundamental science directions. Brief summares of projects and accomplishments for the period for each division are included.« less

Laboratory Directed Research and Development LDRD-FY-2011

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dena Tomchak

2012-03-01

This report provides a summary of the research conducted at the Idaho National Laboratory (INL) during Fiscal Year (FY) 2011. This report demonstrates the types of cutting edge research the INL is performing to help ensure the nation's energy security. The research conducted under this program is aligned with our strategic direction, benefits the Department of Energy (DOE) and is in compliance with DOE order 413.2B. This report summarizes the diverse research and development portfolio with emphasis on the DOE Office of Nuclear Energy (DOE-NE) mission, encompassing both advanced nuclear science and technology and underlying technologies.

Evolution and the Distribution of Glutaminyl and Asparaginyl Residues in Proteins

PubMed Central

Robinson, Arthur B.

1974-01-01

Recent experiments on the deamidation of glutaminyl and asparaginyl residues in peptides and proteins support the hypothesis that these residues may serve as molecular clocks that control biological processes. A hypothesis is now offered that suggests that these molecular clocks are set by rejection or accumulation of appropriate sequences of residues including a glutaminyl or asparaginyl residue during evolution. PMID:4522799

Modeling of microstructure evolution in direct metal laser sintering: A phase field approach

NASA Astrophysics Data System (ADS)

Nandy, Jyotirmoy; Sarangi, Hrushikesh; Sahoo, Seshadev

2017-02-01

Direct Metal Laser Sintering (DMLS) is a new technology in the field of additive manufacturing, which builds metal parts in a layer by layer fashion directly from the powder bed. The process occurs within a very short time period with rapid solidification rate. Slight variations in the process parameters may cause enormous change in the final build parts. The physical and mechanical properties of the final build parts are dependent on the solidification rate which directly affects the microstructure of the material. Thus, the evolving of microstructure plays a vital role in the process parameters optimization. Nowadays, the increase in computational power allows for direct simulations of microstructures during materials processing for specific manufacturing conditions. In this study, modeling of microstructure evolution of Al-Si-10Mg powder in DMLS process was carried out by using a phase field approach. A MATLAB code was developed to solve the set of phase field equations, where simulation parameters include temperature gradient, laser scan speed and laser power. The effects of temperature gradient on microstructure evolution were studied and found that with increase in temperature gradient, the dendritic tip grows at a faster rate.

Sexual selection and the adaptive evolution of PKDREJ protein in primates and rodents.

PubMed

Vicens, Alberto; Gómez Montoto, Laura; Couso-Ferrer, Francisco; Sutton, Keith A; Roldan, Eduardo R S

2015-02-01

PKDREJ is a testis-specific protein thought to be located on the sperm surface. Functional studies in the mouse revealed that loss of PKDREJ has effects on sperm transport and the ability to undergo an induced acrosome reaction. Thus, PKDREJ has been considered a potential target of post-copulatory sexual selection in the form of sperm competition. Proteins involved in reproductive processes often show accelerated evolution. In many cases, this rapid divergence is promoted by positive selection which may be driven, at least in part, by post-copulatory sexual selection. We analysed the evolution of the PKDREJ protein in primates and rodents and assessed whether PKDREJ divergence is associated with testes mass relative to body mass, which is a reliable proxy of sperm competition levels. Evidence of an association between the evolutionary rate of the PKDREJ gene and testes mass relative to body mass was not found in primates. Among rodents, evidence of positive selection was detected in the Pkdrej gene in the family Cricetidae but not in Muridae. We then assessed whether Pkdrej divergence is associated with episodes of sperm competition in these families. We detected a positive significant correlation between the evolutionary rates of Pkdrej and testes mass relative to body mass in cricetids. These findings constitute the first evidence of post-copulatory sexual selection influencing the evolution of a protein that participates in the mechanisms regulating sperm transport and the acrosome reaction, strongly suggesting that positive selection may act on these fertilization steps, leading to advantages in situations of sperm competition. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Evolution of patchily distributed proteins shared between eukaryotes and prokaryotes: Dictyostelium as a case study.

PubMed

Andersson, Jan O

2011-04-01

Protein families are often patchily distributed in the tree of life; they are present in distantly related organisms, but absent in more closely related lineages. This could either be the result of lateral gene transfer between ancestors of organisms that encode them, or losses in the lineages that lack them. Here a novel approach is developed to study the evolution of patchily distributed proteins shared between prokaryotes and eukaryotes. Proteins encoded in the genome of cellular slime mold Dictyostelium discoideum and a restricted number of other lineages, including at least one prokaryote, were identified. Analyses of the phylogenetic distribution of 49 such patchily distributed protein families showed conflicts with organismal phylogenies; 25 are shared with the distantly related amoeboflagellate Naegleria (Excavata), whereas only two are present in the more closely related Entamoeba. Most protein families show unexpected topologies in phylogenetic analyses; eukaryotes are polyphyletic in 85% of the trees. These observations suggest that gene transfers have been an important mechanism for the distribution of patchily distributed proteins across all domains of life. Further studies of this exchangeable gene fraction are needed for a better understanding of the origin and evolution of eukaryotic genes and the diversification process of eukaryotes. Copyright © 2011 S. Karger AG, Basel.

PCR-mediated site-directed mutagenesis.

PubMed

Carey, Michael F; Peterson, Craig L; Smale, Stephen T

2013-08-01

Unlike traditional site-directed mutagenesis, this protocol requires only a single PCR step using full plasmid amplification to generate point mutants. The method can introduce small mutations into promoter sites and is even better suited for introducing single or double mutations into proteins. It is elegant in its simplicity and can be applied quite easily in any laboratory using standard protein expression vectors and commercially available reagents.

Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

PubMed

Ostafe, Raluca; Prodanovic, Radivoje; Nazor, Jovana; Fischer, Rainer

2014-03-20

Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

RNA-Interference Pathways Display High Rates of Adaptive Protein Evolution in Multiple Invertebrates

PubMed Central

Palmer, William H.; Hadfield, Jarrod D.; Obbard, Darren J.

2018-01-01

Conflict between organisms can lead to a reciprocal adaptation that manifests as an increased evolutionary rate in genes mediating the conflict. This adaptive signature has been observed in RNA-interference (RNAi) pathway genes involved in the suppression of viruses and transposable elements in Drosophila melanogaster, suggesting that a subset of Drosophila RNAi genes may be locked in an arms race with these parasites. However, it is not known whether rapid evolution of RNAi genes is a general phenomenon across invertebrates, or which RNAi genes generally evolve adaptively. Here we use population genomic data from eight invertebrate species to infer rates of adaptive sequence evolution, and to test for past and ongoing selective sweeps in RNAi genes. We assess rates of adaptive protein evolution across species using a formal meta-analytic framework to combine data across species and by implementing a multispecies generalized linear mixed model of mutation counts. Across species, we find that RNAi genes display a greater rate of adaptive protein substitution than other genes, and that this is primarily mediated by positive selection acting on the genes most likely to defend against viruses and transposable elements. In contrast, evidence for recent selective sweeps is broadly spread across functional classes of RNAi genes and differs substantially among species. Finally, we identify genes that exhibit elevated adaptive evolution across the analyzed insect species, perhaps due to concurrent parasite-mediated arms races. PMID:29437826

Laboratory evolution of the migratory polymorphism in the sand cricket: combining physiology with quantitative genetics.

PubMed

Roff, Derek A; Fairbairn, Daphne J

2007-01-01

Predicting evolutionary change is the central goal of evolutionary biology because it is the primary means by which we can test evolutionary hypotheses. In this article, we analyze the pattern of evolutionary change in a laboratory population of the wing-dimorphic sand cricket Gryllus firmus resulting from relaxation of selection favoring the migratory (long-winged) morph. Based on a well-characterized trade-off between fecundity and flight capability, we predict that evolution in the laboratory environment should result in a reduction in the proportion of long-winged morphs. We also predict increased fecundity and reduced functionality and weight of the major flight muscles in long-winged females but little change in short-winged (flightless) females. Based on quantitative genetic theory, we predict that the regression equation describing the trade-off between ovary weight and weight of the major flight muscles will show a change in its intercept but not in its slope. Comparisons across generations verify all of these predictions. Further, using values of genetic parameters estimated from previous studies, we show that a quantitative genetic simulation model can account for not only the qualitative changes but also the evolutionary trajectory. These results demonstrate the power of combining quantitative genetic and physiological approaches for understanding the evolution of complex traits.

Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

DOEpatents

Waldo, Geoffrey S.

2007-09-18

The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

Targeting endogenous proteins for degradation through the affinity-directed protein missile system.

PubMed

Fulcher, Luke J; Hutchinson, Luke D; Macartney, Thomas J; Turnbull, Craig; Sapkota, Gopal P

2017-05-01

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel-Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. © 2017 The Authors.

Targeting endogenous proteins for degradation through the affinity-directed protein missile system

PubMed Central

Fulcher, Luke J.; Hutchinson, Luke D.; Macartney, Thomas J.; Turnbull, Craig

2017-01-01

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. PMID:28490657

A Novel Application of Synthetic Biology and Directed Evolution to Engineer Phage-based Antibiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Meiye

The emergence of multiple drug resistant bacteria poses threats to human health, agriculture and food safety. Annually over 100,000 deaths and up to $20 billion loss to the U.S. economy are attributed to multiple drug resistant bacteria. With only four new chemical antibiotics in the drug development pipeline, we are in dire need of new solutions to address the emerging threat of multiple drug resistance. We propose a paradigm-changing approach to address the multi-drug resistant bacteria problem by utilizing Synthetic Biology (SynBio) methodologies to create and evolve “designer” bacteriophages or phages – viruses that specifically infect bacteria – to infectmore » and kill newly emerging pathogenic bacterial strains WITHOUT the need for chemical antibiotics. A major advantage of using phage to combat pathogenic bacteria is that phages can co-evolve with their bacterial host, and Sandia can be the first in the world to establish an industrial scale Synthetic Biology pipeline for phage directed evolution for safe, targeted, customizable solution to bacterial drug resistance. Since there is no existing phage directed evolution effort within or outside of Sandia, this proposal is suitable as a high-risk LDRD effort to create the first pipeline for such an endeavor. The high potential reward nature of this proposal will be the immediate impact in decontamination and restoration of surfaces and infrastructure, with longer term impact in human or animal therapeutics. The synthetic biology and screening approaches will lead to fundamental knowledge of phage/bacteria co-evolution, making Sandia a world leader in directed evolution of bacteriophages.« less

Evolution of double white dwarf binaries undergoing direct-impact accretion: Implications for gravitational wave astronomy

NASA Astrophysics Data System (ADS)

Kremer, Kyle; Breivik, Katelyn; Larson, Shane L.; Kalogera, Vassiliki

2017-01-01

For close double white dwarf binaries, the mass-transfer phenomenon known as direct-impact accretion (when the mass transfer stream impacts the accretor directly rather than forming a disc) may play a pivotal role in the long-term evolution of the systems. In this analysis, we explore the long-term evolution of white dwarf binaries accreting through direct-impact and explore implications of such systems to gravitational wave astronomy. We cover a broad range of parameter space which includes initial component masses and the strength of tidal coupling, and show that these systems, which lie firmly within the LISA frequency range, show strong negative chirps which can last as long as several million years. Detections of double white dwarf systems in the direct-impact phase by detectors such as LISA would provide astronomers with unique ways of probing the physics governing close compact object binaries.

Evolution of sparsity and modularity in a model of protein allostery

NASA Astrophysics Data System (ADS)

Hemery, Mathieu; Rivoire, Olivier

2015-04-01

The sequence of a protein is not only constrained by its physical and biochemical properties under current selection, but also by features of its past evolutionary history. Understanding the extent and the form that these evolutionary constraints may take is important to interpret the information in protein sequences. To study this problem, we introduce a simple but physical model of protein evolution where selection targets allostery, the functional coupling of distal sites on protein surfaces. This model shows how the geometrical organization of couplings between amino acids within a protein structure can depend crucially on its evolutionary history. In particular, two scenarios are found to generate a spatial concentration of functional constraints: high mutation rates and fluctuating selective pressures. This second scenario offers a plausible explanation for the high tolerance of natural proteins to mutations and for the spatial organization of their least tolerant amino acids, as revealed by sequence analysis and mutagenesis experiments. It also implies a faculty to adapt to new selective pressures that is consistent with observations. The model illustrates how several independent functional modules may emerge within the same protein structure, depending on the nature of past environmental fluctuations. Our model thus relates the evolutionary history of proteins to the geometry of their functional constraints, with implications for decoding and engineering protein sequences.

Laboratory Directed Research and Development FY2011 Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Craig, W; Sketchley, J; Kotta, P

2012-03-22

A premier applied-science laboratory, Lawrence Livermore National Laboratory (LLNL) has earned the reputation as a leader in providing science and technology solutions to the most pressing national and global security problems. The LDRD Program, established by Congress at all DOE national laboratories in 1991, is LLNL's most important single resource for fostering excellent science and technology for today's needs and tomorrow's challenges. The LDRD internally directed research and development funding at LLNL enables high-risk, potentially high-payoff projects at the forefront of science and technology. The LDRD Program at Livermore serves to: (1) Support the Laboratory's missions, strategic plan, and foundationalmore » science; (2) Maintain the Laboratory's science and technology vitality; (3) Promote recruiting and retention; (4) Pursue collaborations; (5) Generate intellectual property; and (6) Strengthen the U.S. economy. Myriad LDRD projects over the years have made important contributions to every facet of the Laboratory's mission and strategic plan, including its commitment to nuclear, global, and energy and environmental security, as well as cutting-edge science and technology and engineering in high-energy-density matter, high-performance computing and simulation, materials and chemistry at the extremes, information systems, measurements and experimental science, and energy manipulation. A summary of each project was submitted by the principal investigator. Project summaries include the scope, motivation, goals, relevance to DOE/NNSA and LLNL mission areas, the technical progress achieved in FY11, and a list of publications that resulted from the research. The projects are: (1) Nuclear Threat Reduction; (2) Biosecurity; (3) High-Performance Computing and Simulation; (4) Intelligence; (5) Cybersecurity; (6) Energy Security; (7) Carbon Capture; (8) Material Properties, Theory, and Design; (9) Radiochemistry; (10) High-Energy-Density Science; (11) Laser

Extrapolating non-target risk of Bt crops from laboratory to field.

PubMed

Duan, Jian J; Lundgren, Jonathan G; Naranjo, Steve; Marvier, Michelle

2010-02-23

The tiered approach to assessing ecological risk of insect-resistant transgenic crops assumes that lower tier laboratory studies, which expose surrogate non-target organisms to high doses of insecticidal proteins, can detect harmful effects that might be manifested in the field. To test this assumption, we performed meta-analyses comparing results for non-target invertebrates exposed to Bacillus thuringiensis (Bt) Cry proteins in laboratory studies with results derived from independent field studies examining effects on the abundance of non-target invertebrates. For Lepidopteran-active Cry proteins, laboratory studies correctly predicted the reduced field abundance of non-target Lepidoptera. However, laboratory studies incorporating tri-trophic interactions of Bt plants, herbivores and parasitoids were better correlated with the decreased field abundance of parasitoids than were direct-exposure assays. For predators, laboratory tri-trophic studies predicted reduced abundances that were not realized in field studies and thus overestimated ecological risk. Exposure to Coleopteran-active Cry proteins did not significantly reduce the laboratory survival or field abundance of any functional group examined. Our findings support the assumption that laboratory studies of transgenic insecticidal crops show effects that are either consistent with, or more conservative than, those found in field studies, with the important caveat that laboratory studies should explore all ecologically relevant routes of exposure.

Complexity of Gene Expression Evolution after Duplication: Protein Dosage Rebalancing

PubMed Central

Rogozin, Igor B.

2014-01-01

Ongoing debates about functional importance of gene duplications have been recently intensified by a heated discussion of the “ortholog conjecture” (OC). Under the OC, which is central to functional annotation of genomes, orthologous genes are functionally more similar than paralogous genes at the same level of sequence divergence. However, a recent study challenged the OC by reporting a greater functional similarity, in terms of gene ontology (GO) annotations and expression profiles, among within-species paralogs compared to orthologs. These findings were taken to indicate that functional similarity of homologous genes is primarily determined by the cellular context of the genes, rather than evolutionary history. Subsequent studies suggested that the OC appears to be generally valid when applied to mammalian evolution but the complete picture of evolution of gene expression also has to incorporate lineage-specific aspects of paralogy. The observed complexity of gene expression evolution after duplication can be explained through selection for gene dosage effect combined with the duplication-degeneration-complementation model. This paper discusses expression divergence of recent duplications occurring before functional divergence of proteins encoded by duplicate genes. PMID:25197576

FY2007 Laboratory Directed Research and Development Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Craig, W W; Sketchley, J A; Kotta, P R

The Laboratory Directed Research and Development (LDRD) annual report for fiscal year 2007 (FY07) provides a summary of LDRD-funded projects for the fiscal year and consists of two parts: An introduction to the LDRD Program, the LDRD portfolio-management process, program statistics for the year, and highlights of accomplishments for the year. A summary of each project, submitted by the principal investigator. Project summaries include the scope, motivation, goals, relevance to Department of Energy (DOE)/National Nuclear Security Administration (NNSA) and Lawrence Livermore National Laboratory (LLNL) mission areas, the technical progress achieved in FY07, and a list of publications that resulted frommore » the research in FY07. Summaries are organized in sections by research category (in alphabetical order). Within each research category, the projects are listed in order of their LDRD project category: Strategic Initiative (SI), Exploratory Research (ER), Laboratory-Wide Competition (LW), and Feasibility Study (FS). Within each project category, the individual project summaries appear in order of their project tracking code, a unique identifier that consists of three elements. The first is the fiscal year the project began, the second represents the project category, and the third identifies the serial number of the proposal for that fiscal year.« less

Thermotolerant Yeast Strains Adapted by Laboratory Evolution Show Trade-Off at Ancestral Temperatures and Preadaptation to Other Stresses.

PubMed

Caspeta, Luis; Nielsen, Jens

2015-07-21

A major challenge for the production of ethanol from biomass-derived feedstocks is to develop yeasts that can sustain growth under the variety of inhibitory conditions present in the production process, e.g., high osmolality, high ethanol titers, and/or elevated temperatures (≥ 40 °C). Using adaptive laboratory evolution, we previously isolated seven Saccharomyces cerevisiae strains with improved growth at 40 °C. Here, we show that genetic adaptations to high temperature caused a growth trade-off at ancestral temperatures, reduced cellular functions, and improved tolerance of other stresses. Thermotolerant yeast strains showed horizontal displacement of their thermal reaction norms to higher temperatures. Hence, their optimal and maximum growth temperatures increased by about 3 °C, whereas they showed a growth trade-off at temperatures below 34 °C. Computational analysis of the physical properties of proteins showed that the lethal temperature for yeast is around 49 °C, as a large fraction of the yeast proteins denature above this temperature. Our analysis also indicated that the number of functions involved in controlling the growth rate decreased in the thermotolerant strains compared with the number in the ancestral strain. The latter is an advantageous attribute for acquiring thermotolerance and correlates with the reduction of yeast functions associated with loss of respiration capacity. This trait caused glycerol overproduction that was associated with the growth trade-off at ancestral temperatures. In combination with altered sterol composition of cellular membranes, glycerol overproduction was also associated with yeast osmotolerance and improved tolerance of high concentrations of glucose and ethanol. Our study shows that thermal adaptation of yeast is suitable for improving yeast resistance to inhibitory conditions found in industrial ethanol production processes. Yeast thermotolerance can significantly reduce the production costs of biomass

Chemical evolution of Macondo crude oil during laboratory degradation as characterized by fluorescence EEMs and hydrocarbon composition.

PubMed

Zhou, Zhengzhen; Liu, Zhanfei; Guo, Laodong

2013-01-15

The fluorescence EEM technique, PARAFAC modeling, and hydrocarbon composition were used to characterize oil components and to examine the chemical evolution and degradation pathways of Macondo crude oil under controlled laboratory conditions. Three major fluorescent oil components were identified, with Ex/Em maxima at 226/328, 262/315, and 244/366 nm, respectively. An average degradation half-life of ∼20 d was determined for the oil components based on fluorescence EEM and hydrocarbon composition measurements, showing a dynamic chemical evolution and transformation of the oil during degradation. Dispersants appeared to change the chemical characteristics of oil, to shift the fluorescence EEM spectra, and to enhance the degradation of low-molecular-weight hydrocarbons. Photochemical degradation played a dominant role in the transformation of oil components, likely an effective degradation pathway of oil in the water column. Results from laboratory experiments should facilitate the interpretation of field-data and provide insights for understanding the fate and transport of oil components in the Gulf of Mexico. Copyright © 2012 Elsevier Ltd. All rights reserved.

Molecular evolution of a chordate specific family of G protein-coupled receptors

PubMed Central

2011-01-01

Background Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success. Results We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C) in vertebrates, and a fourth homologue present only in mammals (GPRC5D). Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A in vivo, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling. Conclusions GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non-chordates to become

Laboratory directed research and development annual report 2004.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

This report summarizes progress from the Laboratory Directed Research and Development (LDRD) program during fiscal year 2004. In addition to a programmatic and financial overview, the report includes progress reports from 352 individual R and D projects in 15 categories. The 15 categories are: (1) Advanced Concepts; (2) Advanced Manufacturing; (3) Biotechnology; (4) Chemical and Earth Sciences; (5) Computational and Information Sciences; (6) Differentiating Technologies; (7) Electronics and Photonics; (8) Emerging Threats; (9) Energy and Critical Infrastructures; (10) Engineering Sciences; (11) Grand Challenges; (12) Materials Science and Technology; (13) Nonproliferation and Materials Control; (14) Pulsed Power and High Energy Densitymore » Sciences; and (15) Corporate Objectives.« less

The Evolution of the Secreted Regulatory Protein Progranulin.

PubMed

Palfree, Roger G E; Bennett, Hugh P J; Bateman, Andrew

2015-01-01

Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

Adaptive Evolution of the Venom-Targeted vWF Protein in Opossums that Eat Pitvipers

PubMed Central

Jansa, Sharon A.; Voss, Robert S.

2011-01-01

The rapid evolution of venom toxin genes is often explained as the result of a biochemical arms race between venomous animals and their prey. However, it is not clear that an arms race analogy is appropriate in this context because there is no published evidence for rapid evolution in genes that might confer toxin resistance among routinely envenomed species. Here we report such evidence from an unusual predator-prey relationship between opossums (Marsupialia: Didelphidae) and pitvipers (Serpentes: Crotalinae). In particular, we found high ratios of replacement to silent substitutions in the gene encoding von Willebrand Factor (vWF), a venom-targeted hemostatic blood protein, in a clade of opossums known to eat pitvipers and to be resistant to their hemorrhagic venom. Observed amino-acid substitutions in venom-resistant opossums include changes in net charge and hydrophobicity that are hypothesized to weaken the bond between vWF and one of its toxic snake-venom ligands, the C-type lectin-like protein botrocetin. Our results provide the first example of rapid adaptive evolution in any venom-targeted molecule, and they support the notion that an evolutionary arms race might be driving the rapid evolution of snake venoms. However, in the arms race implied by our results, venomous snakes are prey, and their venom has a correspondingly defensive function in addition to its usual trophic role. PMID:21731638

3D Printing of Protein Models in an Undergraduate Laboratory: Leucine Zippers

ERIC Educational Resources Information Center

Meyer, Scott C.

2015-01-01

An upper-division undergraduate laboratory experiment is described that explores the structure/function relationship of protein domains, namely leucine zippers, through a molecular graphics computer program and physical models fabricated by 3D printing. By generating solvent accessible surfaces and color-coding hydrophobic, basic, and acidic amino…

Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

PubMed

Garcia-Seisdedos, Hector; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

2012-01-01

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous

Probing the Mutational Interplay between Primary and Promiscuous Protein Functions: A Computational-Experimental Approach

PubMed Central

Garcia-Seisdedos, Hector; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

2012-01-01

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous

The temporal evolution of pyroclast ejection velocity and exit trajectory, a laboratory case study.

NASA Astrophysics Data System (ADS)

Cigala, Valeria; Kueppers, Ulrich; José Peña Fernández, Juan; Sesterhenn, Jörn; Taddeucci, Jacopo; Dingwell, Donald Bruce

2017-04-01

. Moreover, the velocity decay was used to investigate the accuracy of the empirical fragmentation depth model from Alatorre-Ibargüengoitia et al. (2011), when different gas-particle ratios are employed. This model is not reproducing all experimental constellations satisfactorily. Further experiments will help to develop this model further. Vent geometry, particle size and temperature show the largest effect on ejection velocity and trajectory. In particular, we observed a positive correlation of velocity with 1) diverging vent walls and 2) temperature and a negative correlation with 1) starting tube length and 2) particle size. On the other hand, exit trajectories show negative correlation with 1) diverging walls, 2) starting tube length, 3) temperature and 4) particle size. Moreover, we found that gas-particle ratio additionally strongly affects the temporal evolution of particle ejection velocity and trajectory. These results highlight the importance of scaled and repeatable laboratory experiments for an enhanced understanding of natural volcanic phenomena that bear direct observability. A closer link will enhance volcanic hazard assessment.

LLL Octopus network: some lessons and future directions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watson, R.W.

1978-06-27

The Octopus network, designed and developed by the Lawrence Livermore Laboratory, is a pioneering, high-performance, local computer network. Several lessons derived from the 14 years of experience in the evolution of Octopus are described, and some of the directions to be taken in its medium-term future are indicated. 3 figures.

Laboratory insights into the chemical and kinetic evolution of several organic molecules under simulated Mars surface UV radiation conditions

NASA Astrophysics Data System (ADS)

Poch, O.; Kaci, S.; Stalport, F.; Szopa, C.; Coll, P.

2014-11-01

The search for organic carbon at the surface of Mars, as clues of past habitability or remnants of life, is a major science goal of Mars' exploration. Understanding the chemical evolution of organic molecules under current martian environmental conditions is essential to support the analyses performed in situ. What molecule can be preserved? What is the timescale of organic evolution at the surface? This paper presents the results of laboratory investigations dedicated to monitor the evolution of organic molecules when submitted to simulated Mars surface ultraviolet radiation (190-400 nm), mean temperature (218 ± 2 K) and pressure (6 ± 1 mbar) conditions. Experiments are done with the MOMIE simulation setup (for Mars Organic Molecules Irradiation and Evolution) allowing both a qualitative and quantitative characterization of the evolution the tested molecules undergo (Poch, O. et al. [2013]. Planet. Space Sci. 85, 188-197). The chemical structures of the solid products and the kinetic parameters of the photoreaction (photolysis rate, half-life and quantum efficiency of photodecomposition) are determined for glycine, urea, adenine and chrysene. Mellitic trianhydride is also studied in order to complete a previous study done with mellitic acid (Stalport, F., Coll, P., Szopa, C., Raulin, F. [2009]. Astrobiology 9, 543-549), by studying the evolution of mellitic trianhydride. The results show that solid layers of the studied molecules have half-lives of 10-103 h at the surface of Mars, when exposed directly to martian UV radiation. However, organic layers having aromatic moieties and reactive chemical groups, as adenine and mellitic acid, lead to the formation of photoresistant solid residues, probably of macromolecular nature, which could exhibit a longer photostability. Such solid organic layers are found in micrometeorites or could have been formed endogenously on Mars. Finally, the quantum efficiencies of photodecomposition at wavelengths from 200 to 250 nm

Do protein crystals nucleate within dense liquid clusters?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maes, Dominique, E-mail: dommaes@vub.ac.be; Vorontsova, Maria A.; Potenza, Marco A. C.

2015-06-27

The evolution of protein-rich clusters and nucleating crystals were characterized by dynamic light scattering (DLS), confocal depolarized dynamic light scattering (cDDLS) and depolarized oblique illumination dark-field microscopy. Newly nucleated crystals within protein-rich clusters were detected directly. These observations indicate that the protein-rich clusters are locations for crystal nucleation. Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10{sup −3} of the solution. According to the two-step mechanism of nucleation, the protein-rich clustersmore » serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein

Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein

PubMed Central

Tan, Wei-Hung; Cheng, Shu-Chun; Liu, Yu-Tung; Wu, Cheng-Guo; Lin, Min-Han; Chen, Chiao-Che; Lin, Chao-Hsiung; Chou, Chi-Yuan

2016-01-01

Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We’ve also identified four historical mutations that are able to produce a “GST-like” S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution. PMID:27499004

LDRD 2016 Annual Report: Laboratory Directed Research and Development Program Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatton, D.

Each year, Brookhaven National Laboratory (BNL) is required to provide a program description and overview of its Laboratory Directed Research and Development Program (LDRD) to the Department of Energy (DOE) in accordance with DOE Order 413.2C dated October 22, 2015. This report provides a detailed look at the scientific and technical activities for each of the LDRD projects funded by BNL in FY 2016, as required. In FY 2016, the BNL LDRD Program funded 48 projects, 21 of which were new starts, at a total cost of $11.5M. The investments that BNL makes in its LDRD program support the Laboratory’smore » strategic goals. BNL has identified four Critical Outcomes that define the Laboratory’s scientific future and that will enable it to realize its overall vision. Two operational Critical Outcomes address essential operational support for that future: renewal of the BNL campus; and safe, efficient laboratory operations.« less

Significant expansion of exon-bordering protein domains during animal proteome evolution

PubMed Central

Liu, Mingyi; Walch, Heiko; Wu, Shaoping; Grigoriev, Andrei

2005-01-01

We present evidence of remarkable genome-wide mobility and evolutionary expansion for a class of protein domains whose borders locate close to the borders of their encoding exons. These exon-bordering domains are more numerous and widely distributed in the human genome than other domains. They also co-occur with more diverse domains to form a larger variety of domain architectures in human proteins. A systematic comparison of nine animal genomes from nematodes to mammals revealed that exon-bordering domains expanded faster than other protein domains in both abundance and distribution, as well as the diversity of co-occurring domains and the domain architectures of harboring proteins. Furthermore, exon-bordering domains exhibited a particularly strong preference for class 1-1 intron phase. Our findings suggest that exon-bordering domains were amplified and interchanged within a genome more often and/or more successfully than other domains during evolution, probably the result of extensive exon shuffling and gene duplication events. The diverse biological functions of these domains underscore the important role they play in the expansion and diversification of animal proteomes. PMID:15640447

Protein-protein interaction network-based detection of functionally similar proteins within species.

PubMed

Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

2012-07-01

Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent. Copyright © 2012 Wiley Periodicals, Inc.

Protein Engineering Approaches in the Post-Genomic Era.

PubMed

Singh, Raushan K; Lee, Jung-Kul; Selvaraj, Chandrabose; Singh, Ranjitha; Li, Jinglin; Kim, Sang-Yong; Kalia, Vipin C

2018-01-01