CiteULike: msuarezdiez's library [68 articles]

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)

A comprehensive library of fluorescent transcriptional reporters for Escherichia coli

Alon Zaslaver — 2006-07-22T19:59:39-00:00

Nature Methods, Vol. 3, No. 8. (21 July 2006), pp. 623-628.

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.

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.

Protein Folding in the Absence of the Solvent Ordering Contribution to the Hydrophobic Interaction

Derek Woolfson — 2008-07-15T15:06:47-00:00

Journal of Molecular Biology, Vol. 229, No. 2. (20 January 1993), pp. 502-511.

Despite considerable effort there is no consensus as to what interaction, or set of interactions, provides the dominant force that drives proteins folding and specifies folded protein structures. A key thermodynamic observation is that a large drop in heat capacity ([Delta]Cp) usually accompanies folding in water. Various factors may contribute to this effect, especially changes in the structure of the solvent upon exposure of both non-polar and polar groups in the unfolded state. The unfavourable Gibbs free energy of solvating non-polar groups, in particular, is thought to provide a central driving force for folding (the hydrophobic effect) but the role of solvent ordering in this remains a matter of controversy. We report here a series of experiments that show that a protein can fold into its native conformation under conditions where solvent ordering effects are demonstrably negligible. In methanol/water mixtures ubiquitin unfolds reversibly with a [Delta]Cp value that falls close to zero above about 30% (v/v) methanol. We are able to reason, on the basis of these data, that the net contribution to the heat capacity change arising primarily from the protein structure itself is not significant and that contributions from changes in solvent ordering are rendered negligible by the change in composition. Nuclear magnetic resonance measurements, however, indicate that non-polar side-chains do still become exposed to solvent in the denatured state under these conditions. The combination of these results and model compound studies suggests that the elimination of ordering effects is an intrinsic properly of the mixed solvent. We can, therefore, conclude that the solvent ordering component of the hydrophobic effect is not an obligatory factor in determining the three dimensional structure into which the protein will fold.

Computational protein design

Christina Kraemer-Pecore — 2008-06-19T09:30:25-00:00

Current Opinion in Chemical Biology, Vol. 5, No. 6. (1 December 2001), pp. 690-695.

The field of computational protein design is reaching its adolescence. Protein design algorithms have been applied to design or engineer proteins that fold, fold faster, catalyze, catalyze faster, signal, and adopt preferred conformational states. Further developments of scoring functions, sampling strategies, and optimization methods will expand the range of applicability of computational protein design to larger and more varied systems, with greater incidence of success. Developments in this field are beginning to have significant impact on biotechnology and chemical biology.

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

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.

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.

Extending Iterative Protein Redesign and Optimization (IPRO) in Protein Library Design for Ligand Specificity

Hossein Fazelinia — 2008-07-23T08:38:40-00:00

Biophys. J., Vol. 92, No. 6. (15 March 2007), pp. 2120-2130.

In this article we extend the Iterative Protein Redesign and Optimization (IPRO) framework for the design of protein libraries with targeted ligand specificity. Mutations that minimize the binding energy with the desired ligand are identified. At the same time explicit constraints are introduced that maintain the binding energy for all decoy ligands above a threshold necessary for successful binding. The proposed framework is demonstrated by computationally altering the effector binding specificity of the bacterial transcriptional regulatory protein AraC, belonging to the AraC/XylS family of transcriptional regulators for different unnatural ligands. The obtained results demonstrate the importance of systematically suppressing the binding energy for competing ligands. Pinpointing a small set of mutations within the binding pocket greatly improves the difference in binding energies between targeted and decoy ligands, even when they are very similar. 10.1529/biophysj.106.096016

Energy Functions for Protein Design: Adjustment with Protein-Protein Complex Affinities, Models for the Unfolded State, and Negative Design of Solubility and Specificity

Navin Pokala — 2008-07-22T11:53:40-00:00

Journal of Molecular Biology, Vol. 347, No. 1. (18 March 2005), pp. 203-227.

The development of the EGAD program and energy function for protein design is described. In contrast to most protein design methods, which require several empirical parameters or heuristics such as patterning of residues or rotamers, EGAD has a minimalist philosophy; it uses very few empirical factors to account for inaccuracies resulting from the use of fixed backbones and discrete rotamers in protein design calculations, and describes the unfolded state, aggregates, and alternative conformers explicitly with physical models instead of fitted parameters. This approach unveils important issues in protein design that are often camouflaged by heuristic-emphasizing methods. Inter-atom energies are modeled with the OPLS-AA all-atom forcefield, electrostatics with the generalized Born continuum model, and the hydrophobic effect with a solvent-accessible surface area-dependent term. Experimental characterization of proteins designed with an unmodified version of the energy function revealed problems with under-packing, stability, aggregation, and structural specificity. Under-packing was addressed by modifying the van der Waals function. By optimizing only three parameters, the effects of >400 mutations on protein-protein complex formation were predicted to within 1.0 kcal mol-1. As an independent test, this modified energy function was used to predict the stabilities of >1500 mutants to within 1.0 kcal mol-1; this required a physical model of the unfolded state that includes more interactions than traditional tripeptide-based models. Solubility and structural specificity were addressed with simple physical approximations of aggregation and conformational equilibria. The complete energy function can design protein sequences that have high levels of identity with their natural counterparts, and have predicted structural properties more consistent with soluble and uniquely folded proteins than the initial designs.

Design of a Hyperstable Protein by Rational Consideration of Unfolded State Interactions

B Anil — 2008-07-22T09:25:17-00:00

J. Am. Chem. Soc., Vol. 128, No. 10. (15 March 2006), pp. 3144-3145.

Abstract: Stabilization of proteins is a long-sought objective. Targeting the unfolded state interactions of a protein is not a method used for this purpose, although many proteins are known to contain such interactions. The N-terminal domain of ribosomal protein L9 (NTL9) has a lysine residue at position 12, which makes strong non-native interactions in the unfolded state. Substitution of a D-alanine for G34 in NTL9 is known to stabilize the protein by reducing the entropy of the unfolded state. Here we combine these two mutations to design a hyperstable protein. The structure of the variant is the same as that of wild-type as judged by 2D NMR. The variant is hyperstable as judged by denaturation experiments, where complete thermal unfolding of the protein does not occur in native buffer.

Role of residual structure in the unfolded state of a thermophilic protein.

S Robic — 2007-03-30T11:22:51-00:00

Proc Natl Acad Sci U S A, Vol. 100, No. 20. (30 September 2003), pp. 11345-11349.

Ribonucleases H from the thermophilic bacterium Thermus thermophilus and the mesophile Escherichia coli demonstrate a dramatic and surprising difference in their change in heat capacity upon unfolding (DeltaCp degrees ). The lower DeltaCp degrees of the thermophilic protein directly contributes to its higher thermal denaturation temperature (Tm). We propose that this DeltaCp degrees difference originates from residual structure in the unfolded state of the thermophilic protein; we verify this hypothesis by using a mutagenic approach. Residual structure in the unfolded state may provide a mechanism for balancing a high Tm with the optimal thermodynamic stability for a protein's function. Structure in the unfolded state is shown to differentially affect the thermodynamic profiles of thermophilic and mesophilic proteins.

Acceptable Protein and Solvent Behavior in Primary Hydration Shell Simulations of Hen Lysozyme

Mehdi Hamaneh — 2007-10-01T09:33:01-00:00

Biophys. J., Vol. 92, No. 7. (1 April 2007), pp. L49-51.

The "primary hydration shell" method in molecular dynamics simulations uses a two- to three-layer thick shell of explicitly represented water molecules as the solvent around the protein of interest. We show that despite its simplicity, this computationally cheap model is capable of predicting acceptable water and protein behavior using the CHARMM22/CMAP potential function. For protein dynamics, comparisons are made with Lipari-Szabo order parameters. These have been derived from NMR relaxation parameters for pico-nano second motions of the NH groups in the main-chain and NH2 groups in Asn/Gln side chains in hen lysozyme. It is also shown that an even simpler, and therefore faster, water-shell model leads to results in similarly good agreement with experiments, and also compared with simulations using a full box of water with periodic boundary conditions or with an implicit solvation model. Thus, the primary hydration shell method should be useful in making larger systems accessible to extensive simulations. 10.1529/biophysj.106.103010

A ?solvated rotamer? approach to modeling water-mediated hydrogen bonds at protein-protein interfaces

Lin Jiang — 2006-06-16T14:00:44-00:00

Proteins: Structure, Function, and Bioinformatics, Vol. 58, No. 4. (2005), pp. 893-904.

Water-mediated hydrogen bonds play critical roles at protein-protein and protein-nucleic acid interfaces, and the interactions formed by discrete water molecules cannot be captured using continuum solvent models. We describe a simple model for the energetics of water-mediated hydrogen bonds, and show that, together with knowledge of the positions of buried water molecules observed in X-ray crystal structures, the model improves the prediction of free-energy changes upon mutation at protein-protein interfaces, and the recovery of native amino acid sequences in protein interface design calculations. We then describe a ?solvated rotamer? approach to efficiently predict the positions of water molecules, at protein-protein interfaces and in monomeric proteins, that is compatible with widely used rotamer-based side-chain packing and protein design algorithms. Finally, we examine the extent to which the predicted water molecules can be used to improve prediction of amino acid identities and protein-protein interface stability, and discuss avenues for overcoming current limitations of the approach. Proteins 2005. © 2005 Wiley-Liss, Inc.

Explicit and implicit water simulations of a &bgr;-hairpin peptide

Buyong Ma — 2008-07-15T16:01:31-00:00

Proteins: Structure, Function, and Genetics, Vol. 37, No. 1. (1999), pp. 73-87.

The conformational properties of a &bgr;-hairpin peptide (YITNSDGTWT) were studied by using both explicit and implicit water simulations. The conformational space of the peptide was scanned by using a restricted hydrogen-bonding search method. The search method used generated the conformational space with enough diversity and good representation of &bgr;-hairpin structures. By using a total surface area-based treatment of hydrophobic interactions, implicit water simulations failed to discriminate between experimental &bgr;-hairpin structures from the rest of the conformers present in the authors' conformation library. However, with inclusion of vibrational free energy and accounting separately for polar and nonpolar surface areas, the nuclear magnetic resonance structure was ranked successfully as the most stable conformation. There is a loose correlation between the conformational energies by the continuum model and the conformational energies by explicit water simulation for conformers with similar structures. However, in terms of solvation energy, both approaches have a much better correlation. By using proper treatment of surface effect (partition of the surface area into polar and nonpolar areas) and including vibrational free-energy contribution, the continuum models should be reliable. Furthermore, the authors found that, for this peptide, &bgr;-hairpin structures have large vibrational entropy that contributes decisively to the stability of folded &bgr;-hairpin structures. Proteins 1999;37:73-87. © 1999 Wiley-Liss, Inc.

Free energy landscape of protein folding in water: Explicit vs. implicit solvent

Ruhong Zhou — 2007-11-03T15:32:21-00:00

Proteins: Structure, Function, and Genetics, Vol. 53, No. 2. (2003), pp. 148-161.

The Generalized Born (GB) continuum solvent model is arguably the most widely used implicit solvent model in protein folding and protein structure prediction simulations; however, it still remains an open question on how well the model behaves in these large-scale simulations. The current study uses the ?-hairpin from C-terminus of protein G as an example to explore the folding free energy landscape with various GB models, and the results are compared to the explicit solvent simulations and experiments. All free energy landscapes are obtained from extensive conformation space sampling with a highly parallel replica exchange method. Because solvation model parameters are strongly coupled with force fields, five different force field/solvation model combinations are examined and compared in this study, namely the explicit solvent model: OPLSAA/SPC model, and the implicit solvent models: OPLSAA/SGB (Surface GB), AMBER94/GBSA (GB with Solvent Accessible Surface Area), AMBER96/GBSA, and AMBER99/GBSA. Surprisingly, we find that the free energy landscapes from implicit solvent models are quite different from that of the explicit solvent model. Except for AMBER96/GBSA, all other implicit solvent models find the lowest free energy state not the native state. All implicit solvent models show erroneous salt-bridge effects between charged residues, particularly in OPLSAA/SGB model, where the overly strong salt-bridge effect results in an overweighting of a non-native structure with one hydrophobic residue F52 expelled from the hydrophobic core in order to make better salt bridges. On the other hand, both AMBER94/GBSA and AMBER99/GBSA models turn the ?-hairpin in to an ?-helix, and the ?-helical content is much higher than the previously reported ?-helices in an explicit solvent simulation with AMBER94 (AMBER94/TIP3P). Only AMBER96/GBSA shows a reasonable free energy landscape with the lowest free energy structure the native one despite an erroneous salt-bridge between D47 and K50. Detailed results on free energy contour maps, lowest free energy structures, distribution of native contacts, ?-helical content during the folding process, NOE comparison with NMR, and temperature dependences are reported and discussed for all five models. Proteins 2003. © 2003 Wiley-Liss, Inc.

Toward Full-Sequence De Novo Protein Design with Flexible Templates for Human Beta-Defensin-2

Ho Fung — 2008-07-08T09:53:46-00:00

Biophys. J., Vol. 94, No. 2. (15 January 2008), pp. 584-599.

In this article, we introduce and apply our de novo protein design framework, which observes true backbone flexibility, to the redesign of human -defensin-2, a 41-residue cationic antimicrobial peptide of the innate immune system. The flexible design templates are generated using molecular dynamics simulations with both Generalized Born implicit solvation and explicit water molecules. These backbone templates were employed in addition to the x-ray crystal structure for designing human -defensin-2. The computational efficiency of our framework was demonstrated with the full-sequence design of the peptide with flexible backbone templates, corresponding to the mutation of all positions except the native cysteines. 10.1529/biophysj.107.110627

Protein design simulations suggest that side-chain conformational entropy is not a strong determinant of amino acid environmental preferences.

X Hu — 2006-02-01T17:31:32-00:00

Proteins, Vol. 62, No. 3. (15 March 2006), pp. 739-748.

Loss of side-chain conformational entropy is an important force opposing protein folding and the relative preferences of the amino acids for being buried or solvent exposed may be partially determined by which amino acids lose more side-chain entropy when placed in the core of a protein. To investigate these preferences, we have incorporated explicit modeling of side-chain entropy into the protein design algorithm, RosettaDesign. In the standard version of the program, the energy of a particular sequence for a fixed backbone depends only on the lowest energy side-chain conformations that can be identified for that sequence. In the new model, the free energy of a single amino acid sequence is calculated by evaluating the average energy and entropy of an ensemble of structures generated by Monte Carlo sampling of amino acid side-chain conformations. To evaluate the impact of including explicit side-chain entropy, sequences were designed for 110 native protein backbones with and without the entropy model. In general, the differences between the two sets of sequences are modest, with the largest changes being observed for the longer amino acids: methionine and arginine. Overall, the identity between the designed sequences and the native sequences does not increase with the addition of entropy, unlike what is observed when other key terms are added to the model (hydrogen bonding, Lennard-Jones energies, and solvation energies). These results suggest that side-chain conformational entropy has a relatively small role in determining the preferred amino acid at each residue position in a protein.

De Novo Protein Design. I. In Search of Stability and Specificity

P Koehl — 2008-06-19T10:42:40-00:00

Journal of Molecular Biology (November 1999), pp. 1161-1181.

We have developed a fully automated protein design strategy that works on the entire sequence of the protein and uses a full atom representation. At each step of the procedure, an all-atom model of the protein is built using the template protein structure and the current designed sequence. The energy of the model is used to drive a Monte Carlo optimization in sequence space: random moves are either accepted or rejected based on the Metropolis criterion. We rely on the physical forces that stabilize native protein structures to choose the optimum sequence. Our energy function includes van der Waals interactions, electrostatics and an environment free energy. Successful protein design should be specific and generate a sequence compatible with the template fold and incompatible with competing folds. We impose specificity by maintaining the amino acid composition constant, based on the random energy model. The specificity of the optimized sequence is tested by fold recognition techniques. Successful sequence designs for the B1 domain of protein G, for the lambda repressor and for sperm whale myoglobin are presented. We show that each additional term of the energy function improves the performance of our design procedure: the van der Waals term ensures correct packing, the electrostatics term increases the specificity for the correct native fold, and the environment solvation term ensures a correct pattern of buried hydrophobic and exposed hydrophilic residues. For the globin family, we show that we can design a protein sequence that is stable in the myoglobin fold, yet incompatible with the very similar hemoglobin fold. Copyright 1999 Academic Press

Energy estimation in protein design

Joaquim Mendes — 2006-08-04T11:56:50-00:00

Current Opinion in Structural Biology, Vol. 12, No. 4. (1 August 2002), pp. 441-446.

The progress achieved by several groups in the field of computational protein design shows that successful design methods include two major features: efficient algorithms to deal with the combinatorial exploration of sequence space and optimal energy functions to rank sequences according to their fitness for the given fold.

Genome-wide prediction of G4 DNA as regulatory motifs: Role in Escherichia coli global regulation

Pooja Rawal — 2007-02-14T15:46:27-00:00

Genome Res., Vol. 16, No. 5. (1 May 2006), pp. 644-655.

The role of nonlinear DNA in replication, recombination, and transcription has become evident in recent years. Although several studies have predicted and characterized regulatory elements at the sequence level, very few have investigated DNA structure as regulatory motifs. Here, using G-quadruplex or G4 DNA motifs as a model, we have researched the role of DNA structure in transcription on a genome-wide scale. Analyses of >61,000 open reading frames (ORFs) across 18 prokaryotes show enrichment of G4 motifs in regulatory regions and indicate its predominance within promoters of genes pertaining to transcription, secondary metabolite biosynthesis, and signal transduction. Based on this, we predict that G4 DNA may present regulatory signals. This is supported by conserved G4 motifs in promoters of orthologous genes across phylogenetically distant organisms. We hypothesized a regulatory role of G4 DNA during supercoiling stress, when duplex destabilization may result in G4 formation. This is in line with our observations from target site analysis for 55 DNA-binding proteins in Escherichia coli, which reveals significant (P < 0.001) association of G4 motifs with target sites of global regulators FIS and Lrp and the sigma factor RpoD (sigma70). These factors together control >1000 genes in the early growth phase and are believed to be induced by supercoiled DNA. We also predict G4 motif-induced supercoiling sensitivity for >30 operons in E. coli, and our findings implicate G4 DNA in DNA-topology-mediated global gene regulation in E. coli. 10.1101/gr.4508806

Anatomy of Escherichia coli sigma70 promoters.

Ryan K Shultzaberger — 2007-01-10T07:35:50-00:00

Nucleic Acids Res (22 December 2006)

Information theory was used to build a promoter model that accounts for the -10, the -35 and the uncertainty of the gap between them on a common scale. Helical face assignment indicated that base -7, rather than -11, of the -10 may be flipping to initiate transcription. We found that the sequence conservation of sigma(70) binding sites is 6.5 +/- 0.1 bits. Some promoters lack a -35 region, but have a 6.7 +/- 0.2 bit extended -10, almost the same information as the bipartite promoter. These results and similarities between the contacts in the extended -10 binding and the -35 suggest that the flexible bipartite sigma factor evolved from a simpler polymerase. Binding predicted by the bipartite model is enriched around 35 bases upstream of the translational start. This distance is the smallest 5' mRNA leader necessary for ribosome binding, suggesting that selective pressure minimizes transcript length. The promoter model was combined with models of the transcription factors Fur and Lrp to locate new promoters, to quantify promoter strengths, and to predict activation and repression. Finally, the DNA-bending proteins Fis, H-NS and IHF frequently have sites within one DNA persistence length from the -35, so bending allows distal activators to reach the polymerase.

Activities of Constitutive Promoters in Escherichia coli

ST Liang — 2008-06-04T11:53:17-00:00

Journal of Molecular Biology (September 1999), pp. 19-37.

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+spoT+strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient relAspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the -lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage ; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of alac deletion strain. The amount of 5-terminal lacZ mRNA and of -galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combininglacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (relAspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity ofrrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+spoT+) relative to ppGpp-deficient (relAspoT) bacterial strains. Copyright 1999 Academic Press

Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool.

JD Sander — 2007-08-26T20:18:42-00:00

Nucleic Acids Res, Vol. 35, No. Web Server issue. (1 July 2007)

Zinc Finger Targeter (ZiFiT) is a simple and intuitive web-based tool that facilitates the design of zinc finger proteins (ZFPs) that can bind to specific DNA sequences. The current version of ZiFiT is based on a widely employed method of ZFP design, the 'modular assembly' approach, in which pre-existing individual zinc fingers are linked together to recognize desired target DNA sequences. Several research groups have described experimentally characterized zinc finger modules that bind many of the 64 possible DNA triplets. ZiFiT leverages the combined capabilities of three of the largest and best characterized module archives by enabling users to select fingers from any of these sets. ZiFiT searches a query DNA sequence for target sites for which a ZFP can be designed using modules available in one or more of the three archives. In addition, ZiFiT output facilitates identification of specific zinc finger modules that are publicly available from the Zinc Finger Consortium. ZiFiT is freely available at http://bindr.gdcb.iastate.edu/ZiFiT/.

Function of the zinc finger in Escherichia coli Fpg protein

J Tchou — 2008-05-19T14:43:08-00:00

J. Biol. Chem., Vol. 268, No. 35. (15 December 1993), pp. 26738-26744.

DksA affects ppGpp induction of RpoS at a translational level.

L Brown — 2006-09-19T05:58:14-00:00

J Bacteriol, Vol. 184, No. 16. (August 2002), pp. 4455-4465.

The RpoS sigma factor (also called sigmaS or sigma38) is known to regulate at least 50 genes in response to environmental sources of stress or during entry into stationary phase. Regulation of RpoS abundance and activity is complex, with many factors participating at multiple levels. One factor is the nutritional stress signal ppGpp. The absence of ppGpp blocks or delays the induction of rpoS during entry into stationary phase. Artificially inducing ppGpp, without starvation, is known to induce rpoS during the log phase 25- to 50-fold. Induction of ppGpp is found to have only minor effects on rpoS transcript abundance or on RpoS protein stability; instead, the efficiency of rpoS mRNA translation is increased by ppGpp as judged by both RpoS pulse-labeling and promoter-independent effects on lacZ fusions. DksA is found to affect RpoS abundance in a manner related to ppGpp. Deleting dksA blocks rpoS induction by ppGpp. Overproduction of DksA induces rpoS but not ppGpp. Deleting dksA neither alters regulation of ppGpp in response to amino acid starvation nor nullifies the inhibitory effects of ppGpp on stable RNA synthesis. Although this suggests that dksA is epistatic to ppGpp, inducing ppGpp does not induce DksA. A dksA deletion does display a subset of the same multiple-amino-acid requirements found for ppGpp(0) mutants, but overproducing DksA does not satisfy ppGpp(0) requirements. Sequenced spontaneous extragenic suppressors of dksA polyauxotrophy are frequently the same T563P rpoB allele that suppresses a ppGpp(0) phenotype. We propose that DksA functions downstream of ppGpp but indirectly regulates rpoS induction.

Zinc-finger motifs expressed in E. coli and folded in vitro direct specific binding to DNA

Kiyoshi Nagai — 2008-05-19T13:39:42-00:00

Nature, Vol. 332, No. 6161. (17 March 1988), pp. 284-286.

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.

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.

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.

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.

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.

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.

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.