Biomedical subjects

Bruce Tidor

Publications and source records attributed to Bruce Tidor.

At least 19 recordsLinked to original sources

PubMed2006

Biological network design strategies: discovery through dynamic optimization.

An important challenge in systems biology is the inherent complexity of biological network models, which complicates the task of relating network structure to function and of understanding the conceptual design principles by which a given network operates. Here we investigate an approach to analyze the relationship between a network structure and its function using the framework of optimization. A common feature found in a variety of biochemical networks involves the opposition of a pair of enzymatic chemical modification reactions such as phosphorylation-dephosphorylation or methylation-demethylation. The modification pair frequently adjusts biochemical properties of its target, such as activating and deactivating function. We applied optimization methodology to study a reversible modification network unit commonly found in signal transduction systems, and we explored the use of this methodology to discover design principles. The results demonstrate that different sets of rate constants used to parameterize the same network topology represent different compromises made in the resulting network operating characteristics. Moreover, the same topology can be used to encode different strategies for achieving performance goals. The ability to adopt multiple strategies may lead to significantly improved performance across a range of conditions through rate modulation or evolutionary processes. The optimization framework explored here is a practical approach to support the discovery of design principles in biological networks.

Computer Simulation↗

PubMed2006

Rational design of new binding specificity by simultaneous mutagenesis of calmodulin and a target peptide.

Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies the role of CaM as a shared participant in calcium-dependent signal transduction pathways but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/target pairs that retain high affinity for one another but lack affinity for wild-type CaM and its natural interaction partners would therefore be useful as sensor components and possibly also as elements of "synthetic" cellular-signaling networks. Here, we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal-muscle light-chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained an affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple-linked binding equilibria.

Amino Acid Sequence↗

PubMed2006

Optimal charges in lead progression: a structure-based neuraminidase case study.

Collective experience in structure-based lead progression has found electrostatic interactions to be more difficult to optimize than shape-based ones. A major reason for this is that the net electrostatic contribution observed includes a significant nonintuitive desolvation component in addition to the more intuitive intermolecular interaction component. To investigate whether knowledge of the ligand optimal charge distribution can facilitate more intuitive design of electrostatic interactions, we took a series of small-molecule influenza neuraminidase inhibitors with known protein cocrystal structures and calculated the difference between the optimal and actual charge distributions. This difference from the electrostatic optimum correlates with the calculated electrostatic contribution to binding (r(2) = 0.94) despite small changes in binding modes caused by chemical substitutions, suggesting that the optimal charge distribution is a useful design goal. Furthermore, detailed suggestions for chemical modification generated by this approach are in many cases consistent with observed improvements in binding affinity, and the method appears to be useful despite discrete chemical constraints. Taken together, these results suggest that charge optimization is useful in facilitating generation of compound ideas in lead optimization. Our results also provide insight into design of neuraminidase inhibitors.

Binding Sites↗

PubMed2005

Quantitative methods for developing Fc mutants with extended half-lives.

Fc mutants with increased binding affinity for the neonatal receptor, FcRn, exhibit increased half-lives in vivo, and represent an attractive means for extending the half-lives of therapeutic antibodies. The half-lives of other therapeutic molecules (e.g., proteins) may also be extended by conjugating them to Fc fragments, thus decreasing the frequency of patient injections and allowing the administration of low and potentially nontoxic concentrations of the therapeutics. To investigate the possibility for further increasing the half-life of Fc, a pair of quantitative methods is presented to complement combinatorial screening and in vivo testing. Specifically, a simple molecular modeling procedure was developed to predict relative Gibbs free energies of binding values (DeltaDeltaGbind) between Fc and FcRn across different mutants and species. This procedure was found to reasonably reproduce experimental DeltaDeltaGbind values from our experiments and the literature, and may be used as an initial screen to explore Fc sequence space more fully prior to experimental testing. In addition, a mathematical model of Fc trafficking was formulated and combined with a cell-level pulse-chase assay to obtain a quantitative recycling parameter in human T84 cells. This Fc recycling parameter was found to be correlated with binding affinity, but captures the pH dependent nature of the interaction between Fc and FcRn and may serve as an additional screen following combinatorial experiments.

Animals↗

PubMed2005

Design of improved protein inhibitors of HIV-1 cell entry: Optimization of electrostatic interactions at the binding interface.

Continuum electrostatic methods are a powerful tool for the analysis and design of biomolecular complexes, with methodologies that allow for the detailed analysis of the electrostatic contributions to binding affinities and procedures for computing the properties of electrostatically optimal ligands. We have applied these methods to the design of improved inhibitors of HIV-1 cell entry. HIV infection of a cell requires viral-cell membrane fusion, an event partially driven by a large-scale conformational change in the viral membrane glycoprotein gp41. This transformation involves the docking of a helix from the C-terminal region of three gp41 chains against a pre-formed trimeric-coiled coil; several protein constructs that inhibit membrane fusion act by binding to an isolated C-terminal helix and blocking the formation of the fusogenic structure. A detailed analysis of the electrostatic contributions to the binding of one such inhibitor (5-Helix) to a C-terminal helix was performed using the X-ray crystal structure of the core of the HIV-1 gp41 ectodomain as a structural model, and several residues on 5-Helix that make substantial contributions to binding, both favorable and unfavorable, were identified. An electrostatic affinity optimization methodology was applied to the side chains of 5-Helix, with the results showing that significant improvements in binding affinity are possible if the electrostatic contributions to the binding free energy are optimized. Several mutations accessible by experimental methods are suggested, with calculated improvements in binding affinity of as much as 500-fold and greater.

Amino Acid Sequence↗

PubMed2005

Interdisciplinary research and education at the biology-engineering-computer science interface: a perspective.

Progress in the life sciences, including genome sequencing and high-throughput experimentation, offers an opportunity for understanding biology and medicine from a systems perspective. This 'new view', which complements the more traditional component-based approach, involves the integration of biological research with approaches from engineering disciplines and computer science. The result is more than a new set of technologies. Rather, it promises a fundamental reconceptualization of the life sciences based on the development of quantitative and predictive models to describe crucial processes. To achieve this change, learning communities are being formed at the interface of the life sciences, engineering and computer science. Through these communities, research and education will be integrated across disciplines and the challenges associated with multidisciplinary team-based science will be addressed.

Biological Science Disciplines↗

PubMed2005

Osmotic pressure of aqueous chondroitin sulfate solution: a molecular modeling investigation.

The osmotic pressure of chondroitin sulfate (CS) solution in contact with an aqueous 1:1 salt reservoir of fixed ionic strength is studied using a recently developed coarse-grained molecular model. The effects of sulfation type (4- vs. 6-sulfation), sulfation pattern (statistical distribution of sulfate groups along a chain), ionic strength, CS intrinsic stiffness, and steric interactions on CS osmotic pressure are investigated. At physiological ionic strength (0.15 M NaCl), the sulfation type and pattern, as measured by a standard statistical description of copolymerization, are found to have a negligible influence on CS osmotic pressure, which depends principally on the mean volumetric fixed charge density. The intrinsic backbone stiffness characteristic of polysaccharides such as CS, however, is demonstrated to contribute significantly to its osmotic pressure behavior, which is similar to that of a solution of charged rods for the 20-disaccharide chains considered. Steric excluded volume is found to play a negligible role in determining CS osmotic pressure at physiological ionic strength due to the dominance of repulsive intermolecular electrostatic interactions that maintain chains maximally spaced in that regime, whereas at high ionic-strength steric interactions become dominant due to electrostatic screening. Osmotic pressure predictions are compared to experimental data and to well-established theoretical models including the Donnan theory and the Poisson-Boltzmann cylindrical cell model.

Chondroitin Sulfates↗

PubMed2005

A coarse-grained molecular model for glycosaminoglycans: application to chondroitin, chondroitin sulfate, and hyaluronic acid.

A coarse-grained molecular model is presented for the study of the equilibrium conformation and titration behavior of chondroitin (CH), chondroitin sulfate (CS), and hyaluronic acid (HA)-glycosaminoglycans (GAGs) that play a central role in determining the structure and biomechanical properties of the extracellular matrix of articular cartilage. Systematic coarse-graining from an all-atom description of the disaccharide building blocks retains the polyelectrolytes' specific chemical properties while enabling the simulation of high molecular weight chains that are inaccessible to all-atom representations. Results are presented for the characteristic ratio, the ionic strength-dependent persistence length, the pH-dependent expansion factor for the end-to-end distance, and the titration behavior of the GAGs. Although 4-sulfation of the N-acetyl-D-galactosamine residue is found to increase significantly the intrinsic stiffness of CH with respect to 6-sulfation, only small differences in the titration behavior of the two sulfated forms of CH are found. Persistence length expressions are presented for each type of GAG using a macroscopic (wormlike chain-based) and a microscopic (bond vector correlation-based) definition. Model predictions agree quantitatively with experimental conformation and titration measurements, which support use of the model in the investigation of equilibrium solution properties of GAGs.

Biophysical Phenomena↗

PubMed2005

Action-at-a-distance interactions enhance protein binding affinity.

The identification of protein mutations that enhance binding affinity may be achieved by computational or experimental means, or by a combination of the two. Sources of affinity enhancement may include improvements to the net balance of binding interactions of residues forming intermolecular contacts at the binding interface, such as packing and hydrogen-bonding interactions. Here we identify noncontacting residues that make substantial contributions to binding affinity and that also provide opportunities for mutations that increase binding affinity of the TEM1 beta-lactamase (TEM1) to the beta-lactamase inhibitor protein (BLIP). A region of BLIP not on the direct TEM1-binding surface was identified for which changes in net charge result in particularly large increases in computed binding affinity. Some mutations to the region have previously been characterized, and our results are in good correspondence with this results of that study. In addition, we propose novel mutations to BLIP that were computed to improve binding significantly without contacting TEM1 directly. This class of noncontacting electrostatic interactions could have general utility in the design and tuning of binding interactions.

Hydrogen Bonding↗

PubMed2005

Defining Cdk5 ligand chemical space with small molecule inhibitors of tau phosphorylation.

Cyclin-dependent kinase 5 (Cdk5) is widely viewed as a possible target for a wide variety of neurological disorders. One pathological role attributed to Cdk5 is the abnormal phosphorylation of tau that may lead to the neuronal inclusions known as neurofibrillary tangles. A high through-put screen for inhibitors of Cdk5-mediated phosphorylation of tau resulted in three compounds with distinct mechanisms of action. One compound is competitive with ATP and has a high affinity for the Cdk5 ATP binding pocket. The second compound also competes with ATP, is noncompetitive with tau, and (uniquely among this class of inhibitors) displaces adjacent amino acid residues to make room for the nitrophenyl group. A third compound did not compete with ATP, but did compete with tau at low concentrations of tau. The SAR and charge optimization derived from cocrystals of the two ATP competitors along with cocrystals of three other ATP competitors map out the importance of filling and properly charging different regions of the ATP binding pocket. Taken together, this analysis shows how the structure of Cdk5 constrains the space of potential inhibitors and reveals a pocket unfilled in all of the structures. These leads could be a starting point for structure-based drug design of more potent and selective inhibitors.

Adenosine Triphosphate↗

PubMed2004

A computational method for the analysis and prediction of protein:phosphopeptide-binding sites.

Phosphopeptide-binding domains, including the FHA, SH2, WW, WD40, MH2, and Polo-box domains, as well as the 14-3-3 proteins, exert control functions in important processes such as cell growth, division, differentiation, and apoptosis. Structures and mechanisms of phosphopeptide binding are generally diverse, revealing few general principles. A computational method for analysis of phosphopeptide-binding domains was therefore developed to elucidate the physical and chemical nature of phosphopeptide binding, given this lack of structural similarity. The surfaces of nine phosphopeptide-binding proteins, representing seven distinct classes of phosphopeptide-binding modules, were discretized, and encoded with information about amino acid identity, surface curvature, and electrostatic potential at every point on the surface in order to identify local surface properties enriched in phosphoresidue contact sites. Cross-validation indicated that propensities corresponding to this enrichment calculated from a subset of the training data could be used to predict the phosphoresidue contact site on proteins not used in training with no false negative results, and with few unconfirmed positive predictions. The locations of phosphoresidue contact sites were then predicted on the surfaces of the checkpoint kinase Chk1 and the BRCA1 BRCT repeat domain, and these predictions are consistent with recent experimental evidence.

Binding Sites↗

PubMed2004

Escherichia coli glutaminyl-tRNA synthetase is electrostatically optimized for binding of its cognate substrates.

Natural evolution has resulted in protein molecules displaying a wide range of binding properties that include extremes of affinity and specificity. A detailed understanding of the principles underlying protein structure-function relationships, particularly with respect to binding properties, would greatly enhance molecular engineering and ligand design studies. Here, we have analyzed the interactions of an aminoacyl-tRNA synthetase for which strong evolutionary pressure has enforced high specificity for substrate binding and catalysis. Electrostatic interactions have been identified as one efficient mechanism for enhancing binding specificity; as such, the effects of charged and polar groups were the focus of this study. The binding of glutaminyl-tRNA synthetase from Escherichia coli to several ligands, including the natural substrates, was analyzed. The electrostatic complementarity of the enzyme to its ligands was assessed using measures derived from affinity optimization theory. The results were independent of the details of the calculational parameters, including the value used for the protein dielectric constant. Glutamine and ATP, two of the natural ligands, were found to be extremely complementary to their binding sites, particularly in regions seen to make electrostatic interactions in the structure. These data suggest that the optimization of electrostatic interactions has played an important role in guiding the evolution of this enzyme. The results also show that the enzyme is able to effectively select for high affinity and specificity for the same chemical moieties both in the context of smaller substrates, and in that of a larger reactive intermediate. The regions of greatest non-complementarity between the enzyme and ligands are the portions of the ligand that make few polar contacts with the binding site, as well as the sites of chemical reaction, where overly strong electrostatic binding interactions with the substrate could hinder catalysis. The results also suggest that the negative charge on the phosphorus center of glutaminyl-adenylate plays an important role in the tight binding of this intermediate, and thus that adenylate analogs that preserve the negative charge in this region may bind substantially tighter than analogs where this group is replaced with a neutral group, such as the sulfamoyl family, which can make similar hydrogen bonds but is uncharged.

Adenosine Monophosphate↗

PubMed2004

Computational and experimental probes of symmetry mismatches in the Arc repressor-DNA complex.

Arc repressor from bacteriophage P22 is a homodimeric member of the ribbon-helix-helix family of transcription factors. Two dimers bind cooperatively to adjacent sites on operator DNA with the antiparallel beta-sheet of each Arc dimer contacting the major groove. Despite the 2-fold symmetry of the dimer, the sequence of the DNA half-site is not symmetric, and thus there is an inherent symmetry mismatch in the complex. Here, using a combination of computational and experimental methods, we further characterize this symmetry mismatch. Continuum electrostatic calculations show that the DNA-contacting side-chains of Gln9, Asn11 and Arg13 each make better electrostatic interactions in one subunit of the Arc dimer than in the other. Based on the computational results, one or both Gln9 side-chains were substituted with Arg or His and Asn11 was similarly substituted with Ile in the context of a single-chain variant of Arc repressor. Although the double mutants that maintain the symmetry of the protein bind much more weakly than scArc, mutants with single Q9R or N11I substitutions bind operator DNA with affinities close to wild-type. Whereas the effects are nearly additive for the mutations at position 9, the effects of single N11I mutations are non-additive. The calculations thus enabled the identification of functionally tolerated mutations in the DNA-binding surface of Arc repressor, which in turn revealed energetic consequences of asymmetric environments in this protein-DNA interaction.

Base Pair Mismatch↗

PubMed2004

X-ray structural and simulation analysis of a protein mutant: the value of a combined approach.

The effect of the mutation Arg 96 to His on the stability of bacteriophage T4 lysozyme has been previously studied by calorimetric experiments, X-ray crystallography, and free energy simulation techniques. The experimental and calculated values for the difference between the free energy of denaturation of the mutant and the wild type are in reasonable agreement. However, the two approaches led to different explanations for the loss in stability. To analyze the differences, a series of refinements based on the crystallographic data were performed, a number of aspects of the simulations were reexamined, and continuum electrostatic calculations were done to complement the latter. The results of those comparisons provide a better understanding of the origin of the free energy difference in this mutant. Furthermore, they show the importance of the combined use of simulations and crystallography for interpreting the effects of mutations on the energetics of the system.

Bacteriophage T4↗

PubMed2003

Surface salt bridges, double-mutant cycles, and protein stability: an experimental and computational analysis of the interaction of the Asp 23 side chain with the N-terminus of the N-terminal domain of the ribosomal protein l9.

Experimental and theoretical double-mutant cycles have been used to investigate a salt bridge in the N-terminal domain of the protein L9. Aspartic acid 23 is the only acidic residue involved in a well-defined pairwise interaction, namely, a partially solvent-exposed salt bridge with the protonated N-terminus of the protein. Mutations were studied in which Asp 23 was substituted by alanine, asparagine, and nitrile alanine. Interactions with the N-terminus were probed by comparisons between proteins with a protonated and acetylated N-terminus. The mutants were all folded, and the structures were unchanged from wild type as judged by CD and 2-D NMR. The coupling free energy between the N-terminus and the side chain of Asp 23 measured through double-mutant cycle analysis was favorable and ranged from -0.7 to -1.7 kcal mol(-)(1), depending upon the set of mutants used. This relatively large coupling free energy for a surface salt bridge likely arises from geometric factors that reduce the entropy loss associated with salt-bridge formation and from structural relaxation in the mutants. Coupling free energies computed with continuum electrostatic calculations agreed well with the experimental values when full account was taken of all potential interactions, particularly those involving Asp 23 and the acetylated N-terminus as well as interactions with solvent. The measured and calculated coupling free energy decreased only slightly when the salt concentration was increased from 100 to 750 mM NaCl. The calculations suggest that the coupling free energy between D23 and the N-terminus measured through the experimental double-mutant cycle analysis is significantly smaller than the actual interaction free energy between the groups in the wild-type structure because of the inapplicability of assumptions frequently used to interpret double-mutant cycles.

Amino Acid Substitution↗

PubMed2003

Expression of N-formylated proteins in Escherichia coli.

In bacteria, protein expression initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. In addition, because deformylation is a prerequisite for removal of the initiating methionine, this post-translational processing step is also prevented by actinonin, and the N-formyl methionine residue is retained by proteins from which it is normally removed. We have demonstrated the applicability of this system for obtaining N-modified forms of several different proteins and use one of these modified molecules to show that the N-terminal amino group is not required for ClpXP degradation of proteins bearing an N-terminal recognition signal.

Amidohydrolases↗

PubMed2003

Limitations of quantitative gene regulation models: a case study.

Understanding the relationship between network structure and behavior is fundamental to the field of computational and systems biology. A particularly important distinction is the extent to which qualitative aspects of network performance are encoded in network topology as opposed to being determined through quantitative details, such as those of kinetics. Here, we develop a general and rigorous mathematical framework for the analysis of genetic networks and apply it to a family of synthetic gene networks. A key feature of our methodology involves determining network behavior that is insensitive to kinetic parameters such as rate constants and nonlinear functional dependencies of rates on molecular concentrations. Results indicate that behavior observed in some networks cannot be reconciled with standard gene expression and regulation models. We explore relaxing model assumptions to explain the observed behavior, allowing for both dynamic and stochastic phenomena, and propose an alternative model. Our alternative model includes the suggestion of a new mechanism by which the counterintuitive behavior could be achieved; central to the model is the assumption that the Clp protein degradation system, which is responsible for the regulatory proteins used in this study, becomes saturated.

Computational Biology↗

PubMed2003

Subsystem identification through dimensionality reduction of large-scale gene expression data.

The availability of parallel, high-throughput biological experiments that simultaneously monitor thousands of cellular observables provides an opportunity for investigating cellular behavior in a highly quantitative manner at multiple levels of resolution. One challenge to more fully exploit new experimental advances is the need to develop algorithms to provide an analysis at each of the relevant levels of detail. Here, the data analysis method non-negative matrix factorization (NMF) has been applied to the analysis of gene array experiments. Whereas current algorithms identify relationships on the basis of large-scale similarity between expression patterns, NMF is a recently developed machine learning technique capable of recognizing similarity between subportions of the data corresponding to localized features in expression space. A large data set consisting of 300 genome-wide expression measurements of yeast was used as sample data to illustrate the performance of the new approach. Local features detected are shown to map well to functional cellular subsystems. Functional relationships predicted by the new analysis are compared with those predicted using standard approaches; validation using bioinformatic databases suggests predictions using the new approach may be up to twice as accurate as some conventional approaches.

Algorithms↗