CiteULike: msuarezdiez's library [43 articles]

Insights into the molecular recognition of the 5'-GNN-3' family of DNA sequences by zinc finger domains

Birgit Dreier — 2008-05-15T09:45:30-00:00

Journal of Molecular Biology, Vol. 303, No. 4. (3 November 2000), pp. 489-502.

In order to construct zinc finger domains that recognize all of the possible 64 DNA triplets, it is necessary to understand the mechanisms of protein/DNA interactions on the molecular level. Previously we reported 16 zinc finger domains which had been characterized in detail to bind specifically to the 5'-GNN-3' family of DNA sequences. Artificial transcription factors constructed from these domains can regulate the expression of endogenous genes. These domains were created by phage-display selection followed by site-directed mutagenesis. A total of 84 mutants of a three-domain zinc finger protein have been analyzed for their DNA-binding specificity. Here, we report the results of this systematic and extensive mutagenesis study. New insights into zinc finger/DNA interactions were obtained by combining specificity data with computer modeling and comparison with known structural data from NMR and crystallographic studies. This analysis suggests that unusual cross-strand and inter-helical contacts are made by some of these proteins, and the general orientation of the recognition helix to the DNA is flexible, even when constrained by flanking zinc finger domains. These findings disfavor the utility of existing simple recognition codes and suggest that highly specific domains cannot be obtained from phage display alone in most cases, but only in combination with rational design. The molecular basis of zinc finger/DNA interaction is complex and its understanding is dependent on the analysis of a large number of proteins. This understanding should enable us to refine rapidly the specificity of other zinc finger domains, as well as polydactyl proteins constructed with these domains to recognize extended DNA sequences.

Toward rules relating zinc finger protein sequences and DNA binding site preferences.

JR Desjarlais — 2006-11-06T15:34:38-00:00

Proc Natl Acad Sci U S A, Vol. 89, No. 16. (15 August 1992), pp. 7345-7349.

Zinc finger proteins of the Cys2-His2 type consist of tandem arrays of domains, where each domain appears to contact three adjacent base pairs of DNA through three key residues. We have designed and prepared a series of variants of the central zinc finger within the DNA binding domain of Sp1 by using information from an analysis of a large data base of zinc finger protein sequences. Through systematic variations at two of the three contact positions (underlined), relatively specific recognition of sequences of the form 5'-GGGGN(G or T)GGG-3' has been achieved. These results provide the basis for rules that may develop into a code that will allow the design of zinc finger proteins with preselected DNA site specificity.

Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences.

DJ Segal — 2008-05-15T08:52:40-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, No. 6. (16 March 1999), pp. 2758-2763.

We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5'-GNN-3'. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make base-specific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions -1, 3, and 6 of a zinc finger domain are insufficient to allow for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5'-(GNN)6-3' family of DNA sequences. These materials and methods should allow for the rapid construction of novel gene switches and provide the basis for a universal system for gene control.

A synthetic oscillatory network of transcriptional regulators.

MB Elowitz — 2004-11-22T00:17:30-00:00

Nature, Vol. 403, No. 6767. (20 January 2000), pp. 335-338.

Networks of interacting biomolecules carry out many essential functions in living cells, but the 'design principles' underlying the functioning of such intracellular networks remain poorly understood, despite intensive efforts including quantitative analysis of relatively simple systems. Here we present a complementary approach to this problem: the design and construction of a synthetic network to implement a particular function. We used three transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, termed the repressilator, in Escherichia coli. The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells. The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation. This artificial clock displays noisy behaviour, possibly because of stochastic fluctuations of its components. Such 'rational network design may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks.

Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements.

R Lutz — 2007-02-19T18:42:42-00:00

Nucleic Acids Res, Vol. 25, No. 6. (15 March 1997), pp. 1203-1210.

Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter PLtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.

De novo protein design using pairwise potentials and a genetic algorithm

DT Jones — 2008-04-29T09:22:40-00:00

Protein Sci, Vol. 3, No. 4. (1 April 1994), pp. 567-574.

The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors.

JS Marvin — 2008-04-25T09:19:27-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 9. (29 April 1997), pp. 4366-4371.

Rational protein design is an emerging approach for testing general theories of structure and function. The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value. Here we use the design approach to test the current understanding of the structural principles of allosteric interactions in proteins and demonstrate how a simple allosteric system can form the basis for the construction of a generic biosensor molecular engineering system. We have identified regions in Escherichia coli maltose-binding protein that are predicted to be allosterically linked to its maltose-binding site. Environmentally sensitive fluorophores were covalently attached to unique thiols introduced by cysteine mutations at specific sites within these regions. The fluorescence of such conjugates changes cooperatively with respect to maltose binding, as predicted. Spatial separation of the binding site and reporter groups allows the intrinsic properties of each to be manipulated independently. Provided allosteric linkage is maintained, ligand binding can therefore be altered without affecting transduction of the binding event by fluorescence. To demonstrate applicability to biosensor technology, we have introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand. These mutant proteins have been combined in a composite biosensor capable of measuring substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude.

Recent advances in the bioremediation of persistent organic pollutants via biomolecular engineering

Ee Ang — 2008-04-25T09:04:53-00:00

Enzyme and Microbial Technology, Vol. 37, No. 5. (3 October 2005), pp. 487-496.

With recent advances in biomolecular engineering, the bioremediation of persistent organic pollutants (POPs) using genetically modified microorganisms has become a rapidly growing area of research for environmental protection. Two main biomolecular approaches, rational design and directed evolution, have been developed to engineer enhanced microorganisms and enzymes for the biodegradation of POPs. This review describes the most recent developments and applications of these biomolecular tools for enhancing the capability of microorganisms to bioremediate three major classes of POPs - polycyclic aromatic hydrocabons (PAHs), polychlorinated biphenyls (PCBs) and pesticides. Most of the examples focused on the redesign of various features of the enzymes involved in the bioremediation of POPs, including the enzyme expression level, enzymatic activity and substrate specificity. Overall, the rapidly expanding potential of biomolecular engineering techniques has created the exciting potential of remediating some of the most recalcitrant and hazardous compounds in the environment.

Protein structure prediction by global optimization of a potential energy function

Adam Liwo — 2008-04-24T16:37:18-00:00

Proceedings of the National Academy of Sciences, Vol. 96, No. 10. (11 May 1999), pp. 5482-5485.

10.1073/pnas.96.10.5482

Optimal Sequence Selection in Proteins of Known Structure by Simulated Evolution

HW Hellinga — 2008-04-22T11:48:36-00:00

Proceedings of the National Academy of Sciences, Vol. 91, No. 13. (21 June 1994), pp. 5803-5807.

10.1073/pnas.91.13.5803

Enhancing catalytic promiscuity for biocatalysis.

RJ Kazlauskas — 2007-03-02T07:55:32-00:00

Curr Opin Chem Biol, Vol. 9, No. 2. (April 2005), pp. 195-201.

Catalytic promiscuity - the ability of a single active site to catalyse more than one chemical transformation - has a natural role in evolution and occasionally in biosynthesis of secondary metabolites. Catalytic promiscuity is more widespread than often recognized. Recent success in adding and enhancing such catalytic activities by protein engineering suggests new potential applications in enzyme-catalyzed organic synthesis.

Automatic sequence design of MHC Class-I binding peptides impairing CD8+ T cell recognition

Koji Ogata — 2008-04-22T10:50:59-00:00

J. Biol. Chem. (30 October 2002), M206853200.

An automatic protein design procedure is used to compute amino acid sequences of peptides likely to bind the HLA-A2 MHC class-I allele. The only information used by the procedure are a structural template, a rotamer library, and a well-established classical empirical force field. The calculations are performed on 6 different templates from x-ray structures of HLA-A0201-peptide complexes. Each template consists of the bound peptide backbone and the full atomic coordinates of the MHC protein. Sequences within 2 kcal/mol of the minimum energy sequence are computed for each template and the sequences from all he templates are combined and ranked by their energies. The 5 lowest energy peptide sequences and 5 other low energy sequences re-ranked on the basis of their similarity to peptides known to bind the same MHC allele, are chemically synthesised and tested for their ability to bind and form stable complexes with the HLA-A2 molecule. The most efficient binders are also tested for inhibition of the T-cell receptor (TCR) recognition of two known CD8+ T effectors. Results show that all 10 peptides bind the expected MHC protein. The 6 strongest binders also form stable HLA-A2-peptide complexes, albeit to varying degrees, and 3 peptides display significant inhibition of CD8+ T cell recognition. These results are rationalised in light of our knowledge of the 3D structures of the HLA-A2-peptide and HLA-A2-peptide-TCR complexes. 10.1074/jbc.M206853200

Engineered antibody Fc variants with enhanced effector function.

GA Lazar — 2006-05-01T16:44:11-00:00

Proc Natl Acad Sci U S A, Vol. 103, No. 11. (14 March 2006), pp. 4005-4010.

Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.

De Novo Computational Design of Retro-Aldol Enzymes

Lin Jiang — 2008-03-07T02:58:37-00:00

Science, Vol. 319, No. 5868. (7 March 2008), pp. 1387-1391.

The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model. 10.1126/science.1152692

Kemp elimination catalysts by computational enzyme design

Daniela Röthlisberger — 2008-03-21T04:33:18-00:00

Nature (19 March 2008)

Rapid, accurate, computational discovery of Rho-independent transcription terminators illuminates their relationship to DNA uptake

Carleton Kingsford — 2007-02-22T18:48:09-00:00

Genome Biology, Vol. 8 (21 February 2007), R22.

Enzyme promiscuity: mechanism and applications

Karl Hult — 2007-04-12T09:16:30-00:00

Trends in Biotechnology, Vol. In Press, Corrected Proof

Introductory courses in biochemistry teach that enzymes are specific for their substrates and the reactions they catalyze. Enzymes diverging from this statement are sometimes called promiscuous. It has been suggested that relaxed substrate and reaction specificities can have an important role in enzyme evolution; however, enzyme promiscuity also has an applied aspect. Enzyme condition promiscuity has, for a long time, been used to run reactions under conditions of low water activity that favor ester synthesis instead of hydrolysis. Together with enzyme substrate promiscuity, it is exploited in numerous synthetic applications, from the laboratory to industrial scale. Furthermore, enzyme catalytic promiscuity, where enzymes catalyze accidental or induced new reactions, has begun to be recognized as a valuable research and synthesis tool. Exploiting enzyme catalytic promiscuity might lead to improvements in existing catalysts and provide novel synthesis pathways that are currently not available.

Directed evolution of a (beta alpha)8-barrel enzyme to catalyze related reactions in two different metabolic pathways.

C Jürgens — 2008-03-31T13:53:03-00:00

Proc Natl Acad Sci U S A, Vol. 97, No. 18. (29 August 2000), pp. 9925-9930.

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (betaalpha)(8)-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (betaalpha)(8)-barrel enzymes are very suitable for the design of new catalytic activities.

Engineering a multipurpose catalyst.

RJ Kazlauskas — 2006-11-13T09:19:27-00:00

Nat Chem Biol, Vol. 2, No. 10. (October 2006), pp. 514-515.

New formulae for folding catalysts make them multi-purpose enzymes

Mireille Moutiez — 2008-03-31T13:05:24-00:00

Biotechnology and Bioengineering, Vol. 56, No. 6. (1997), pp. 645-649.

Whereas protein disulfide isomerase (PDI) and prolyl isomerase (PPI) are considered as efficient protein folding catalysts, very few large scale processes use them because of economical and technical limitations. PDI and PPI were successfully immobilized on cross-linked agarose beads. PDI inactivation during coupling reaction was overcome by oxidizing active site thiols with dimethylsulfoxide and led to a 64% active enzyme. Alternatively, PPI and PDI biotinylation resulted in 100% and 55-66% active enzymes respectively. The use of these modified catalysts suppresses post-refolding purification and enables the design of biochemical reactors. Several other possible applications are also discussed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 645-649, 1997.

De novo design of catalytic proteins.

J Kaplan — 2006-03-31T10:55:07-00:00

Proc Natl Acad Sci U S A, Vol. 101, No. 32. (10 August 2004), pp. 11566-11570.

The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function, while simultaneously laying the groundwork for the design of novel catalysts. Here we describe the design of an O(2)-dependent phenol oxidase whose structure, sequence, and activity are designed from first principles. The protein catalyzes the two-electron oxidation of 4-aminophenol (k(cat)/K(M) = 1,500 M(-1).min(-1)) to the corresponding quinone monoimine by using a diiron cofactor. The catalytic efficiency is sensitive to changes of the size of a methyl group in the protein, illustrating the specificity of the design.

Creation of a Broad-Range and Highly Stereoselective D-Amino Acid Dehydrogenase for the One-Step Synthesis of D-Amino Acids

K Vedha-Peters — 2008-03-31T12:36:42-00:00

J. Am. Chem. Soc., Vol. 128, No. 33. (23 August 2006), pp. 10923-10929.

Abstract: Using both rational and random mutagenesis, we have created the first known broad substrate range, nicotinamide cofactor dependent, and highly stereoselective D-amino acid dehydrogenase. This new enzyme is capable of producing D-amino acids via the reductive amination of the corresponding 2-keto acid with ammonia. This biocatalyst was the result of three rounds of mutagenesis and screening performed on the enzyme meso-diaminopimelate D-dehydrogenase. The first round targeted the active site of the wild-type enzyme and produced mutants that were no longer strictly dependent on the native substrate. The second and third rounds produced mutants that had an increased substrate range including straight- and branched-aliphatic amino acids and aromatic amino acids. The very high selectivity toward the D-enantiomer (95 to >99% ee) was shown to be preserved even after the addition of the five mutations found in the three rounds of mutagenesis and screening. This new enzyme could complement and improve upon current methods for D-amino acid synthesis.

Computational Protein Design: A Novel Path to Future Protein Drugs

M Rosenberg — 2008-03-27T12:24:05-00:00

pp. 3973-3997.

Computational protein design emerges in recent years as a field that could make a substantial impact on the design of protein drugs. It still consists mainly of redesigning parts of a protein sequence for increasing the stability of a given 3-dimensional conformation of a protein, but has already been extended from redesigning core residues to redesigning in all other protein regions, as well as to the design of backbone conformations. More recently, proteins with new binding functions and new enzymes, protein libraries, designs of full folds and of a new protein fold, have been some of the main highlights. The search and the scoring problems are however not fully solved, and many of the design processes should be examined on much larger scales in order to assess their usefulness. We examine some of the basic assumptions in computational protein design, in particular, the separation between sequence and scaffold designs. Among others, we suggest to include more protein residues in computations, to include relevant parts of the backbone, to use appropriate reference states, to produce the proteins and to validate the designs by structural examination of the protein products.

Advances in computational protein design

Sheldon Park — 2006-04-10T08:04:10-00:00

Current Opinion in Structural Biology, Vol. 14, No. 4. (August 2004), pp. 487-494.

Computational protein design continues to experience a variety of methodological advances. Several improvements have been suggested for the objective functions used to quantify sequence/structure compatibility. Disparate design strategies based upon dead-end elimination, simulated annealing and statistical design have each recently yielded striking successes involving de novo designed proteins with sizes on the order of 100 residues or greater. Such methods may be used to design new proteins, as well as to redesign natural proteins to facilitate structural and biophysical studies.

Review: Protein Design--Where We Were, Where We Are, Where We're Going

Navin Pokala — 2007-02-10T12:17:27-00:00

Journal of Structural Biology, Vol. 134, No. 2-3. (May 2001), pp. 269-281.

Protein design has become a powerful approach for understanding the relationship between amino acid sequence and 3-dimensional structure. In the past 5 years, there have been many breakthroughs in the development of computational methods that allow the selection of novel sequences given the structure of a protein backbone. Successful design of protein scaffolds has now paved the way for new endeavors to design function. The ability to design sequences compatible with a fold may also be useful in structural and functional genomics by expanding the range of proteins used for fold recognition and for the identification of functionally important domains from multiple sequence alignments.

Modeling mutations in protein structures

Eric Feyfant — 2007-09-04T09:01:24-00:00

Protein Sci, Vol. 16, No. 9. (1 September 2007), pp. 2030-2041.

We describe an automated method for the modeling of point mutations in protein structures. The protein is represented by all non-hydrogen atoms. The scoring function consists of several types of physical potential energy terms and homology-derived restraints. The optimization method implements a combination of conjugate gradient minimization and molecular dynamics with simulated annealing. The testing set consists of 717 pairs of known protein structures differing by a single mutation. Twelve variations of the scoring function were tested in three different environments of the mutated residue. The best-performing protocol optimizes all the atoms of the mutated residue, with respect to a scoring function that includes molecular mechanics energy terms for bond distances, angles, dihedral angles, peptide bond planarity, and non-bonded atomic contacts represented by Lennard-Jones potential, dihedral angle restraints derived from the aligned homologous structure, and a statistical potential for non-bonded atomic interactions extracted from a large set of known protein structures. The current method compares favorably with other tested approaches, especially when predicting long and flexible side-chains. In addition to the thoroughness of the conformational search, sampled degrees of freedom, and the scoring function type, the accuracy of the method was also evaluated as a function of the flexibility of the mutated side-chain, the relative volume change of the mutated residue, and its residue type. The results suggest that further improvement is likely to be achieved by concentrating on the improvement of the scoring function, in addition to or instead of increasing the variety of sampled conformations. 10.1110/ps.072855507

Energy functions for protein design

Benjamin Gordon — 2008-03-26T16:21:43-00:00

Current Opinion in Structural Biology, Vol. 9, No. 4. (August 1999), pp. 509-513.

Recent successes in protein design have illustrated the promise of computational approaches. These methods rely on energy expressions to evaluate the quality of different amino acid sequences for target protein structures. The force fields optimized for design differ from those typically used in molecular mechanics and molecular dynamics calculations.

Coupling backbone flexibility and amino acid sequence selection in protein design.

A Su — 2008-03-26T15:48:56-00:00

Protein Sci, Vol. 6, No. 8. (August 1997), pp. 1701-1707.

Using a protein design algorithm that considers side-chain packing quantitatively, the effect of explicit backbone motion on the selection of amino acids in protein design was assessed in the core of the streptococcal protein G beta 1 domain (G beta 1). Concerted backbone motion was introduced by varying G beta 1's supersecondary structure parameter values. The stability and structural flexibility of seven of the redesigned proteins were determined experimentally and showed that core variants containing as many as 6 of 10 possible mutations retain native-like properties. This result demonstrates that backbone flexibility can be combined explicitly with amino acid side-chain selection and that the selection algorithm is sufficiently robust to tolerate perturbations as large as 15% of G beta 1's native supersecondary structure parameter values.

Progress in computational protein design.

Shaun M Lippow — 2007-08-06T07:16:04-00:00

Curr Opin Biotechnol (17 July 2007)

Current progress in computational structure-based protein design is reviewed in the areas of methodology and applications. Foundational advances include new potential functions, more efficient ways of computing energetics, flexible treatments of solvent, and useful energy function approximations, as well as ensemble-based approaches to scoring designs for inclusion of entropic effects, improvements to guaranteed and to stochastic search techniques, and methods to design combinatorial libraries for screening and selection. Applications include new approaches and successes in the design of specificity for protein folding, binding, and catalysis, in the redesign of proteins for enhanced binding affinity, and in the application of design technology to study and alter enzyme catalysis. Computational protein design continues to mature and advance.

A novel method for accurate operon predictions in all sequenced prokaryotes

Morgan Price — 2005-03-05T12:30:19-00:00

Nucleic Acids Research, Vol. 33, No. 3. (2005), pp. 880-892.

A multi-approaches-guided genetic algorithm with application to operon prediction

Shuqin Wang — 2008-03-24T12:01:12-00:00

Artificial Intelligence in Medicine, Vol. 41, No. 2. (October 2007), pp. 151-159.

SummaryObjective The prediction of operons is critical to the reconstruction of regulatory networks at the whole genome level. Multiple genome features have been used for predicting operons. However, multiple genome features are usually dealt with using only single method in the literatures. The aim of this paper is to develop a combined method for operon prediction by using different methods to preprocess different genome features in order for exerting their unique characteristics.Methods A novel multi-approach-guided genetic algorithm for operon prediction is presented. We exploit different methods for intergenic distance, cluster of orthologous groups (COG) gene functions, metabolic pathway and microarray expression data. A novel local-entropy-minimization method is proposed to partition intergenic distance. Our program can be used for other newly sequenced genomes by transferring the knowledge that has been obtained from Escherichia coli data. We calculate the log-likelihood for COG gene functions and Pearson correlation coefficient for microarray expression data. The genetic algorithm is used for integrating the four types of data.Results The proposed method is examined on E. coli K12 genome, Bacillus subtilis genome, and Pseudomonas aeruginosa PAO1 genome. The accuracies of prediction for these three genomes are 85.9987%, 88.296%, and 81.2384%, respectively.Conclusion Simulated experimental results demonstrate that in the genetic algorithm the preprocessing for genome data using multiple approaches ensures the effective utilization of different biological characteristics. Experimental results also show that the proposed method is applicable for predicting operons in prokaryote.

Operon prediction for sequenced bacterial genomes without experimental information.

NH Bergman — 2008-03-24T11:58:25-00:00

Appl Environ Microbiol, Vol. 73, No. 3. (February 2007), pp. 846-854.

Various computational approaches have been proposed for operon prediction, but most algorithms rely on experimental or functional data that are only available for a small subset of sequenced genomes. In this study, we explored the possibility of using phylogenetic information to aid in operon prediction, and we constructed a Bayesian hidden Markov model that incorporates comparative genomic data with traditional predictors, such as intergenic distances. The prediction algorithm performs as well as the best previously reported method, with several significant advantages. It uses fewer data sources and so it is easier to implement, and the method is more broadly applicable than previous methods--it can be applied to essentially every gene in any sequenced bacterial genome. Furthermore, we show that near-optimal performance is easily reached with a generic set of comparative genomes and does not depend on a specific relationship between the subject genome and the comparative set. We applied the algorithm to the Bacillus anthracis genome and found that it successfully predicted all previously verified B. anthracis operons. To further test its performance, we chose a predicted operon (BA1489-92) containing several genes with little apparent functional relatedness and tested their cotranscriptional nature. Experimental evidence shows that these genes are cotranscribed, and the data have interesting implications for B. anthracis biology. Overall, our findings show that this algorithm is capable of highly sensitive and accurate operon prediction in a wide range of bacterial genomes and that these predictions can lead to the rapid discovery of new functional relationships among genes.

Predicted transcription factor binding sites as predictors of operons in Escherichia coli and Streptomyces coelicolor.

Emma Laing — 2008-02-12T13:39:47-00:00

BMC Genomics, Vol. 9 (12 February 2008), 79.

Operon prediction by comparative genomics: an application to the Synechococcus sp. WH8102 genome.

X Chen — 2005-05-09T20:47:13-00:00

Nucleic Acids Res, Vol. 32, No. 7. (2004), pp. 2147-2157.

We present a computational method for operon prediction based on a comparative genomics approach. A group of consecutive genes is considered as a candidate operon if both their gene sequences and functions are conserved across several phylogenetically related genomes. In addition, various supporting data for operons are also collected through the application of public domain computer programs, and used in our prediction method. These include the prediction of conserved gene functions, promoter motifs and terminators. An apparent advantage of our approach over other operon prediction methods is that it does not require many experimental data (such as gene expression data and pathway data) as input. This feature makes it applicable to many newly sequenced genomes that do not have extensive experimental information. In order to validate our prediction, we have tested the method on Escherichia coli K12, in which operon structures have been extensively studied, through a comparative analysis against Haemophilus influenzae Rd and Salmonella typhimurium LT2. Our method successfully predicted most of the 237 known operons. After this initial validation, we then applied the method to a newly sequenced and annotated microbial genome, Synechococcus sp. WH8102, through a comparative genome analysis with two other cyanobacterial genomes, Prochlorococcus marinus sp. MED4 and P.marinus sp. MIT9313. Our results are consistent with previously reported results and statistics on operons in the literature.

Operons in Escherichia coli: Genomic analyses and predictions

Heladia Salgado — 2008-03-24T11:50:07-00:00

Proceedings of the National Academy of Sciences, Vol. 97, No. 12. (June 2000), pp. 6652-6657.

The rich knowledge of operon organization in Escherichia coli, together with the completed chromosomal sequence of this bacterium, enabled us to perform an analysis of distances between genes and of functional relationships of adjacent genes in the same operon, as opposed to adjacent genes in different transcription units. We measured and demonstrated the expected tendencies of genes within operons to have much shorter intergenic distances than genes at the borders of transcription units. A clear peak at short distances between genes in the same operon contrasts with a flat frequency distribution of genes at the borders of transcription units. Also, genes in the same operon tend to have the same physiological functional class. The results of these analyses were used to implement a method to predict the genomic organization of genes into transcription units. The method has a maximum accuracy of 88% correct identification of pairs of adjacent genes to be in an operon, or at the borders of transcription units, and correctly identifies around 75% of the known transcription units when used to predict the transcription unit organization of the E. coli genome. Based on the frequency distance distributions, we estimated a total of 630 to 700 operons in E. coli. This step opens the possibility of predicting operon organization in other bacteria whose genome sequences have been finished.

Recent advances in computational promoter analysis in understanding the transcriptional regulatory network

Ping Qiu — 2008-03-07T12:55:03-00:00

Biochemical and Biophysical Research Communications, Vol. 309, No. 3. (26 September 2003), pp. 495-501.

The computational approach to the study of transcriptional regulation networks has become more attractive and feasible with the rapid accumulation of complete genome sequences and the advance of high-throughput expression profiling technology. In this review, current computational approaches for understanding the transcriptional regulatory network, including promoter prediction, transcription factor binding site identification, combinatorial regulatory elements prediction, and transcription factor target gene identification, are discussed. The role of comparative genomics in transcription regulatory region analysis is also reviewed.

Detailed analysis of grid-based molecular docking: A case study of CDOCKER - A CHARMm-based MD docking algorithm

Guosheng Wu — 2006-06-14T00:11:40-00:00

Journal of Computational Chemistry, Vol. 24, No. 13. (2003), pp. 1549-1562.

The influence of various factors on the accuracy of protein-ligand docking is examined. The factors investigated include the role of a grid representation of protein-ligand interactions, the initial ligand conformation and orientation, the sampling rate of the energy hyper-surface, and the final minimization. A representative docking method is used to study these factors, namely, CDOCKER, a molecular dynamics (MD) simulated-annealing-based algorithm. A major emphasis in these studies is to compare the relative performance and accuracy of various grid-based approximations to explicit all-atom force field calculations. In these docking studies, the protein is kept rigid while the ligands are treated as fully flexible and a final minimization step is used to refine the docked poses. A docking success rate of 74% is observed when an explicit all-atom representation of the protein (full force field) is used, while a lower accuracy of 66-76% is observed for grid-based methods. All docking experiments considered a 41-member protein-ligand validation set. A significant improvement in accuracy (76 vs. 66%) for the grid-based docking is achieved if the explicit all-atom force field is used in a final minimization step to refine the docking poses. Statistical analysis shows that even lower-accuracy grid-based energy representations can be effectively used when followed with full force field minimization. The results of these grid-based protocols are statistically indistinguishable from the detailed atomic dockings and provide up to a sixfold reduction in computation time. For the test case examined here, improving the docking accuracy did not necessarily enhance the ability to estimate binding affinities using the docked structures. © 2003 Wiley Periodicals, Inc. J Comput Chem 13: 1549-1562, 2003

Monte Carlo simulations of biomolecules: The MC module in CHARMM.

J Hu — 2008-01-16T12:44:03-00:00

J Comput Chem, Vol. 27, No. 2. (30 January 2006), pp. 203-216.

We describe the implementation of a general and flexible Monte Carlo (MC) module for the program CHARMM, which is used widely for modeling biomolecular systems with empirical energy functions. Construction and use of an almost arbitrary move set with only a few commands is made possible by providing several predefined types of moves that can be combined. Sampling can be enhanced by noncanonical acceptance criteria, automatic optimization of step sizes, and energy minimization. A systematic procedure for improving MC move sets is introduced and applied to simulations of two peptides. The resulting move sets allow MC to sample the configuration spaces of these systems much more rapidly than Langevin dynamics. The rate of convergence of the difference in free energy between ethane and methanol in explicit solvent is also examined, and comparable performances are observed for MC and the Nosé-Hoover algorithm. Its ease of use combined with its sampling efficiency make the MC module in CHARMM an attractive alternative for exploring the behavior of biomolecular systems.

Interfacing Q-Chem and CHARMM to perform QM/MM reaction path calculations

Lee Woodcock — 2008-01-16T12:42:26-00:00

Journal of Computational Chemistry, Vol. 28, No. 9. (2007), pp. 1485-1502.

A hybrid quantum mechanical/molecular mechanical (QM/MM) potential energy function with Hartree-Fock, density functional theory (DFT), and post-HF (RIMP2, MP2, CCSD) capability has been implemented in the CHARMM and Q-Chem software packages. In addition, we have modified CHARMM and Q-Chem to take advantage of the newly introduced replica path and the nudged elastic band methods, which are powerful techniques for studying reaction pathways in a highly parallel (i.e., parallel/parallel) fashion, with each pathway point being distributed to a different node of a large cluster. To test our implementation, a series of systems were studied and comparisons were made to both full QM calculations and previous QM/MM studies and experiments. For instance, the differences between HF, DFT, MP2, and CCSD QM/MM calculations of H2O···H2O, H2O···Na+, and H2O···Cl- complexes have been explored. Furthermore, the recently implemented polarizable Drude water model was used to make comparisons to the popular TIP3P and TIP4P water models for doing QM/MM calculations. We have also computed the energetic profile of the chorismate mutase catalyzed Claisen rearrangement at various QM/MM levels of theory and have compared the results with previous studies. Our best estimate for the activation energy is 8.20 kcal/mol and for the reaction energy is -23.1 kcal/mol, both calculated at the MP2/6-31+G(d)//MP2/6-31+G(d)/C22 level of theory. © 2007 Wiley Periodicals, Inc. J Comput Chem, 2007

Studying Protein Folding on the Grid: Experiences using CHARMM on NPACI Resources under Legion

A Natrajan — 2008-01-16T12:38:46-00:00

(2001)

One of the benefits of a computational grid is the ability to run high-performance applications over distributed resources simply and securely. We demonstrated this benefit with an experiment in which we studied the protein-folding process with the CHARMM molecular simulation package over a grid managed by a grid operating system, Legion. High-performance applications can take advantage of grid resources if the grid operating system provides both low-level functionality as well as high-level...

Computational sidechain placement and protein mutagenesis with implicit solvent models.

A Lopes — 2008-01-15T14:52:02-00:00

Proteins, Vol. 67, No. 4. (1 June 2007), pp. 853-867.

Structure prediction and computational protein design should benefit from accurate solvent models. We have applied implicit solvent models to two problems that are central to this area. First, we performed sidechain placement for 29 proteins, using a solvent model that combines a screened Coulomb term with an Accessible Surface Area term (CASA model). With optimized parameters, the prediction quality is comparable with earlier work that omitted electrostatics and solvation altogether. Second, we computed the stability changes associated with point mutations involving ionized sidechains. For over 1000 mutations, including many fully or partly buried positions, we compared CASA and two generalized Born models (GB) with a more accurate model, which solves the Poisson equation of continuum electrostatics numerically. CASA predicts the correct sign and order of magnitude of the stability change for 81% of the mutations, compared to 97% with the best GB. We also considered 140 mutations for which experimental data are available. Comparing to experiment requires additional assumptions about the unfolded protein structure, protein relaxation in response to the mutations, and contributions from the hydrophobic effect. With a simple, commonly-used unfolded state model, the mean unsigned error is 2.1 kcal/mol with both CASA and the best GB. Overall, the electrostatic model is not important for sidechain placement; CASA and GB are equivalent for surface mutations, while GB is far superior for fully or partly buried positions. Thus, for problems like protein design that involve all these aspects, the most recent GB models represent an important step forward. Along with the recent discovery of efficient, pairwise implementations of GB, this will open new possibilities for the computational engineering of proteins.

Neutral genetic drift can alter promiscuous protein functions, potentially aiding functional evolution

Jesse Bloom — 2007-06-29T02:41:16-00:00

Biology Direct, Vol. 2 (28 June 2007), 17.

Distinct Roles for Arp2/3 Regulators in Actin Assembly and Endocytosis

Brian Galletta — 2008-01-14T14:33:26-00:00

PLoS Biology, Vol. 6, No. 1. (1 January 2008), e1.

The Arp2/3 complex is essential for actin assembly and motility in many cell processes, and a large number of proteins have been found to bind and regulate it in vitro. A critical challenge is to understand the actions of these proteins in cells, especially in settings where multiple regulators are present. In a systematic study of the sequential multicomponent actin assembly processes that accompany endocytosis in yeast, we examined and compared the roles of WASp, two type-I myosins, and two other Arp2/3 activators, along with that of coronin, which is a proposed inhibitor of Arp2/3. Quantitative analysis of high-speed fluorescence imaging revealed individual functions for the regulators, manifested in part by novel phenotypes. We conclude that Arp2/3 regulators have distinct and overlapping roles in the processes of actin assembly that drive endocytosis in yeast. The formation of the endocytic actin patch, the creation of the endocytic vesicle, and the movement of the vesicle into the cytoplasm display distinct dependencies on different Arp2/3 regulators. Knowledge of these roles provides insight into the in vivo relevance of the dendritic nucleation model for actin assembly.