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At least 343 records · Page 19Linked to original sources
PubMed2005

Directed evolution of proteins for heterologous expression and stability.

Recent developments have been made in the application of directed evolution to achieve the efficient heterologous expression of proteins in Escherichia coli and yeast by increasing the stability and solubility of the protein in the host environment. One interesting conclusion that emerges is that the evolutionary process often improves the stability and solubility of an intermediate (apoprotein, proprotein or folding intermediate) that otherwise constitutes a bottleneck to functional expression, rather than altering the protein's final state.

Cloning, Molecular↗
PubMed2002

Evolutionary domain fusion expanded the substrate specificity of the transmembrane electron transporter DsbD.

Modular organization of proteins has been postulated as a widely used strategy for protein evolution. The multidomain transmembrane protein DsbD catalyzes the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli. Most bacterial species do not have DsbD, but instead their genomes encode a much smaller protein, CcdA, which resembles the central hydrophobic domain of DsbD. We used reciprocal heterologous complementation assays between E.coli and Rhodobacter capsulatus to show that, despite their differences in size and structure, DsbD and CcdA are functional homologs. While DsbD transfers reducing potential to periplasmic protein disulfide bond isomerases and to the cytochrome c thioreduction pathway, CcdA appears to be involved only in cytochrome c biogenesis. Our findings strongly suggest that, by the acquisition of additional thiol-redox active domains, DsbD expanded its substrate specificity.

Bacterial Proteins↗
PubMed2000

Contrasting patterns of nonneutral evolution in proteins encoded in nuclear and mitochondrial genomes.

We report that patterns of nonneutral DNA sequence evolution among published nuclear and mitochondrially encoded protein-coding loci differ significantly in animals. Whereas an apparent excess of amino acid polymorphism is seen in most (25/31) mitochondrial genes, this pattern is seen in fewer than half (15/36) of the nuclear data sets. This differentiation is even greater among data sets with significant departures from neutrality (14/15 vs. 1/6). Using forward simulations, we examined patterns of nonneutral evolution using parameters chosen to mimic the differences between mitochondrial and nuclear genetics (we varied recombination rate, population size, mutation rate, selective dominance, and intensity of germ line bottleneck). Patterns of evolution were correlated only with effective population size and strength of selection, and no single genetic factor explains the empirical contrast in patterns. We further report that in Arabidopsis thaliana, a highly self-fertilizing plant with effectively low recombination, five of six published nuclear data sets also exhibit an excess of amino acid polymorphism. We suggest that the contrast between nuclear and mitochondrial nonneutrality in animals stems from differences in rates of recombination in conjunction with a distribution of selective effects. If the majority of mutations segregating in populations are deleterious, high linkage may hinder the spread of the occasional beneficial mutation.

Animals↗
PubMed2002

Roles of mutation and recombination in the evolution of protein thermodynamics.

We present a comprehensive study of the evolutionary origin of the thermodynamic behavior of proteins. With the use of a simplified model, we exhaustively enumerate the space of all sequences and the space of all structures, simulate the evolutionary relationship between sequences and structures, and characterize the steady-state sequence distribution for all structures in terms of several thermodynamic variables. We assess the effects of two major forces of evolution: mutation and recombination. Three simplifications are made. First, a two-dimensional lattice model is used to represent protein sequences and structures. Second, proteins undergo neutral evolution so that the fitness landscape has a flat allowed region inside of which all sequences are equally fit. Third, we ignore otherwise important factors such as finite population size and evolutionary time. Two scenarios emerge from our study. The first occurs when evolution is dominated by mutation events. Even though the prototype sequence that is most mutationally robust is preferred by evolution, the preference is not strong enough to offset the huge size of sequence space. Most native sequences are located near the boundary of the fitness region and are marginally compatible with the native structure. The second scenario occurs when evolution is dominated by recombination events. Now evolutionary preference for prototype sequence is strong enough to overcome the size of sequence space so that most native sequences are located near the center of sequence-structure compatibility. We conclude that the relative frequency of mutation and recombination events is a major determinant of how optimal protein sequences are for their structures.

Evolution, Molecular↗
PubMed1999

DNA-binding proteins and evolution of transcription regulation in the archaea.

Likely DNA-binding domains in archaeal proteins were analyzed using sequence profile methods and available structural information. It is shown that all archaea encode a large number of proteins containing the helix-turn-helix (HTH) DNA-binding domains whose sequences are much more similar to bacterial HTH domains than to eukaryotic ones, such as the PAIRED, POU and homeodomains. The predominant class of HTH domains in archaea is the winged-HTH domain. The number and diversity of HTH domains in archaea is comparable to that seen in bacteria. The HTH domain in archaea combines with a variety of other domains that include replication system components, such as MCM proteins, translation system components, such as the alpha-subunit of phenyl-alanyl-tRNA synthetase, and several metabolic enzymes. The majority of the archaeal HTH-containing proteins are predicted to be gene/operon-specific transcriptional regulators. This apparent bacterial-type mode of transcription regulation is in sharp contrast to the eukaryote-like layout of the core transcription machinery in the archaea. In addition to the predicted bacterial-type transcriptional regulators, the HTH domain is conserved in archaeal and eukaryotic core transcription factors, such as TFIIB, TFIIE-alpha and MBF1. MBF1 is the only highly conserved, classical HTH domain that is vertically inherited in all archaea and eukaryotes. In contrast, while eukaryotic TFIIB and TFIIE-alpha possess forms of the HTH domain that are divergent in sequence, their archaeal counterparts contain typical HTH domains. It is shown that, besides the HTH domain, archaea encode unexpectedly large numbers of two other predicted DNA-binding domains, namely the Arc/MetJ domain and the Zn-ribbon. The core transcription regulators in archaea and eukaryotes (TFIIB/TFB, TFIIE-alpha and MBF1) and in bacteria (the sigma factors) share no similarity beyond the presence of distinct HTH domains. Thus HTH domains might have been independently recruited for a role in transcription regulation in the bacterial and archaeal/eukaryotic lineages. During subsequent evolution, the similarity between archaeal and bacterial gene/operon transcriptional regulators might have been established and maintained through multiple horizontal gene transfer events.

Archaea↗
PubMed2005

Folding and stability of a primitive protein.

We have previously attempted to simulate domain creation in early protein evolution by recombining polypeptide segments from non-homologous proteins, and we have described the structure of one such de novo protein, 1b11, a segment-swapped tetramer with novel architecture. Here, we have analyzed the thermodynamic stability and folding kinetics of the 1b11 tetramer and its monomeric and dimeric intermediates, and of 1b11 mutants with changes at the domain interface. Denatured 1b11 polypeptides fold into transient, folded monomers with marginal stability (DeltaG<1kcalmol(-1)) which convert rapidly ( approximately 6x10(4)M(-1)s(-1)) into dimers (DeltaG=9.8kcal/mol) and then more slowly ( approximately 3M(-1)s(-1)) into tetramers (DeltaG=28kcalmol(-1)). Segment swapping takes place during dimerization, as suggested by mass spectroscopic analysis of covalently linked peptides derived from proteolysis of a disulfide-linked dimer. Our results confirm that segment swapping and associated oligomerization are both powerful ways of stabilizing proteins, and we suggest that this may have been a feature of early protein evolution.

Amino Acid Sequence↗
PubMed2004

Directed evolution of protein enzymes using nonhomologous random recombination.

We recently reported the development of nonhomologous random recombination (NRR) as a method for nucleic acid diversification and applied NRR to the evolution of DNA aptamers. Here, we describe a modified method, protein NRR, that enables proteins to access diversity previously difficult or impossible to generate. We investigated the structural plasticity of protein folds and the ability of helical motifs to function in different contexts by applying protein NRR and in vivo selection to the evolution of chorismate mutase (CM) enzymes. Functional CM mutants evolved using protein NRR contained many insertions, deletions, and rearrangements. The distribution of these changes was not random but clustered in certain regions of the protein. Topologically rearranged but functional enzymes also emerged from these studies, indicating that multiple connectivities can accommodate a functional CM active site and demonstrating the ability to generate new domain connectivities through protein NRR. Protein NRR was also used to randomly recombine CM and fumarase, an unrelated but also alpha-helical protein. Whereas the resulting library contained fumarase fragments in many contexts before functional selection, library members surviving selection for CM activity invariably contained a CM core with fumarase sequences found only at the termini or in one loop. These results imply that internal helical fragments cannot be swapped between these proteins without the loss of nearly all CM activity. Our findings suggest that protein NRR will be useful in probing the functional requirements of enzymes and in the creation of new protein topologies.

Chorismate Mutase↗
PubMed1983

Linear repetitions of amino acids and convergent evolution inside protein subregions of ordered secondary structures.

51 polypeptides of known 3-dimensional structures have been submitted to a search for internal similarities. It is shown that the frequency of proteins displaying significant amounts of internal similarities is higher than predicted by chance. A non-negligible part of those similarities probably occurs in connection with the existence of ordered secondary structures. Indeed, similarity occurs at a much more important rate when analyses are restricted to protein subsequences corresponding to alpha helices or beta pleated sheets. Furthermore, the correlation existing between the rates at which linear and inverted repeats occur inside protein subregions of ordered secondary structures suggests that a significant part of short similarities are analogies rather than homologies. An hypothesis is put forward suggesting that the regular alternations of hydrophobicity which characterize most of alpha helices and beta strands could provoke the occurrence of significant amounts of similarities inside protein sequences.

Amino Acid Sequence↗
PubMed2001

Determination of protein function, evolution and interactions by structural genomics.

The genome sequencing projects and knowledge of the entire protein repertoires of many organisms have prompted new procedures and techniques for the large-scale determination of protein structure, function and interactions. Recently, new work has been carried out on the determination of the function and evolutionary relationships of proteins by experimental structural genomics, and the discovery of protein-protein interactions by computational structural genomics.

Evolution, Molecular↗
PubMed2003

In vitro evolution of proteins for drug development.

Thanks to biotechnology, proteins are becoming increasingly important tools to fight disease, both as therapeutics in their own right and as catalysts for the synthesis of small molecule drugs. However, the properties of these proteins are not necessarily optimal for their intended tasks. In vitro evolution is a set of technologies useful to address their shortcomings. Moreover, in vitro evolution can help illuminate natural evolutionary pathways, thus potentially enabling prediction of drug resistance evolution. We consider here recent developments in the area of in vitro evolution, as well as its application to proteins of interest to medical science.

Biological Assay↗
PubMed2002

Swaps in protein sequences.

An important question in protein evolution is to what extent proteins may have undergone swaps (switches of domain or fragment order) during evolution. Such events might have occurred in several forms: Swaps of short fragments, swaps of structural and functional motifs, or recombination of domains in multidomain proteins. This question is important for the theoretical understanding of the evolution of proteins, and has practical implications for using swaps as a design tool in protein engineering. In order to analyze the question systematically, we conducted a large scale survey of possible swaps and permutations among all pairs of protein from the Swissport database. A swap is defined as a specific kind of sequence mutation between two proteins in which two fragments that appear in both sequences have different relative order in the two sequences. For example, aXbYc and dYeXf are defined as a swap, where X and Y represent sequence fragments that switched their order. Identifying such swaps is difficult using standard sequence comparison packages. One of the main problems in the analysis stems from the fact that many sequences contain repeats, which may be identified as false-positive swaps. We have used two different approaches to detect pairs of proteins with swaps. The first approach is based on the predefined list of domains in Pfam. We identified all the proteins that share at least two domains and analyzed their relative order, looking for pairs in which the order of these domains was switched. We designed an algorithm to distinguish between real swaps and duplications. In the second approach, we used Blast to detect pairs of proteins that share several fragments. Then, we used an automatic procedure to select pairs that are likely to contain swaps. Those pairs were analyzed visually, using a graphical tool, to eliminate duplications. Combining these approaches, about 140 different cases of swaps in the Swissprot database were found (after eliminating multiple pairs within the same family). Some of the cases have been described in the literature, but many are novel examples. Although each new example identified may be interesting to analyze, our main conclusion is that cases of swaps are rare in protein evolution. This observation is at odds with the common view that proteins are very modular to the point that modules (e.g., domains) can be shuffled between proteins with minimal constraints. Our study suggests that sequential constraints, i.e., the relative order between domains, are highly conserved.

Algorithms↗
PubMed2000

Helical membrane protein folding, stability, and evolution.

Helical membrane protein folding and oligomerization can be usefully conceptualized as involving two energetically distinct stages-the formation and subsequent side-to-side association of independently stable transbilayer helices. The interactions of helices with the bilayer, with prosthetic groups, and with each other are examined in the context of recent evidence. We conclude that the two-stage concept remains useful as an approach to simplifying discussions of stability, as a framework for folding concepts, and as a basis for understanding membrane protein evolution.

Animals↗