Biomedical subjects

Ivet Bahar

Publications and source records attributed to Ivet Bahar.

At least 19 recordsLinked to original sources

PubMed2006

Predisposition of the dark state of rhodopsin to functional changes in structure.

As the only member of the family of G-protein-coupled receptors for which atomic coordinates are available, rhodopsin is widely studied for insight into the molecular mechanism of G-protein-coupled receptor activation. The currently available structures refer to the inactive, dark state, of rhodopsin, rather than the light-activated metarhodopsin II (Meta II) state. A model for the Meta II state is proposed here by analyzing elastic network normal modes in conjunction with experimental data. Key mechanical features and interactions broken/formed in the proposed model are found to be consistent with the experimental data. The model is further tested by using a set of Meta II fluorescence decay rates measured to empirically characterize the deactivation of rhodopsin mutants. The model is found to correctly predict 93% of the experimentally observed effects in 119 rhodopsin mutants for which the decay rates and misfolding data have been measured, including a systematic analysis of Cys-->Ser replacements reported here. Based on the detailed comparison between model and experiments, a cooperative activation mechanism is deduced that couples retinal isomerization to concerted changes in conformation, facilitated by the intrinsic dynamics of rhodopsin. A global hinge site is identified near the retinal-binding pocket that ensures the efficient propagation of signals from the central transmembrane region to both cytoplasmic and extracellular ends. The predicted activation mechanism opens the transmembrane helices at the critical G-protein binding cytoplasmic domain. This model provides a detailed, mechanistic description of the activation process, extending experimental observations and yielding new insights for further tests.

Algorithms↗

PubMed2006

Topological basis of signal integration in the transcriptional-regulatory network of the yeast, Saccharomyces cerevisiae.

BACKGROUND: Signal recognition and information processing is a fundamental cellular function, which in part involves comprehensive transcriptional regulatory (TR) mechanisms carried out in response to complex environmental signals in the context of the cell's own internal state. However, the network topological basis of developing such integrated responses remains poorly understood. RESULTS: By studying the TR network of the yeast Saccharomyces cerevisiae we show that an intermediate layer of transcription factors naturally segregates into distinct subnetworks. In these topological units transcription factors are densely interlinked in a largely hierarchical manner and respond to external signals by utilizing a fraction of these subnets. CONCLUSION: As transcriptional regulation represents the 'slow' component of overall information processing, the identified topology suggests a model in which successive waves of transcriptional regulation originating from distinct fractions of the TR network control robust integrated responses to complex stimuli.

Algorithms↗

PubMed2006

Anisotropic network model: systematic evaluation and a new web interface.

MOTIVATION: The Anisotropic Network Model (ANM) is a simple yet powerful model for normal mode analysis of proteins. Despite its broad use for exploring biomolecular collective motions, ANM has not been systematically evaluated to date. A lack of a convenient interface has been an additional obstacle for easy usage. RESULTS: ANM has been evaluated on a large set of proteins to establish the optimal model parameters that achieve the highest correlation with experimental data and its limits of accuracy and applicability. Residue fluctuations in globular proteins are shown to be more accurately predicted than those in nonglobular proteins, and core residues are more accurately described than solvent-exposed ones. Significant improvement in agreement with experiments is observed with increase in the resolution of the examined structure. A new server for ANM calculations is presented, which offers flexible options for controlling model parameters and output formats, interactive animation of collective modes and advanced graphical features. AVAILABILITY: ANM server (http://www.ccbb.pitt.edu/anm)

Algorithms↗

PubMed2006

Markov propagation of allosteric effects in biomolecular systems: application to GroEL-GroES.

We introduce a novel approach for elucidating the potential pathways of allosteric communication in biomolecular systems. The methodology, based on Markov propagation of 'information' across the structure, permits us to partition the network of interactions into soft clusters distinguished by their coherent stochastics. Probabilistic participation of residues in these clusters defines the communication patterns inherent to the network architecture. Application to bacterial chaperonin complex GroEL-GroES, an allostery-driven structure, identifies residues engaged in intra- and inter-subunit communication, including those acting as hubs and messengers. A number of residues are distinguished by their high potentials to transmit allosteric signals, including Pro33 and Thr90 at the nucleotide-binding site and Glu461 and Arg197 mediating inter- and intra-ring communication, respectively. We propose two most likely pathways of signal transmission, between nucleotide- and GroES-binding sites across the cis and trans rings, which involve several conserved residues. A striking observation is the opposite direction of information flow within cis and trans rings, consistent with negative inter-ring cooperativity. Comparison with collective modes deduced from normal mode analysis reveals the propensity of global hinge regions to act as messengers in the transmission of allosteric signals.

Allosteric Regulation↗

PubMed2006

oGNM: online computation of structural dynamics using the Gaussian Network Model.

An assessment of the equilibrium dynamics of biomolecular systems, and in particular their most cooperative fluctuations accessible under native state conditions, is a first step towards understanding molecular mechanisms relevant to biological function. We present a web-based system, oGNM that enables users to calculate online the shape and dispersion of normal modes of motion for proteins, oligonucleotides and their complexes, or associated biological units, using the Gaussian Network Model (GNM). Computations with the new engine are 5-6 orders of magnitude faster than those using conventional normal mode analyses. Two cases studies illustrate the utility of oGNM. The first shows that the thermal fluctuations predicted for 1250 non-homologous proteins correlate well with X-ray crystallographic data over a broad range [7.3-15 A] of inter-residue interaction cutoff distances and the correlations improve with increasing observation temperatures. The second study, focused on 64 oligonucleotides and oligonucleotide-protein complexes, shows that good agreement with experiments is achieved by representing each nucleotide by three GNM nodes (as opposed to one-node-per-residue in proteins) along with uniform interaction ranges for all components of the complexes. These results open the way to a rapid assessment of the dynamics of DNA/RNA-containing complexes. The server can be accessed at http://ignm.ccbb.pitt.edu/GNM_Online_Calculation.htm.

Algorithms↗

PubMed2006

Common mechanism of pore opening shared by five different potassium channels.

A fundamental question associated with the function of ion channels is the conformational changes that allow for reversibly opening/occluding the pore through which the cations permeate. The recently elucidated crystal structures of potassium channels reveal similar structural motifs at their pore-forming regions, suggesting that they share a common gating mechanism. The validity of this hypothesis is explored by analyzing the collective dynamics of five known K(+) channel structures. Normal-mode analysis using the Gaussian network model strikingly reveals that all five structures display the same intrinsic motions at their pore-forming region despite the differences in their sequences, structures, and activation mechanisms. Superposition of the most cooperative mode profiles shows that the identified common mechanism is a global corkscrew-like counterrotation of the extracellular and cytoplasmic (CP) regions, leading to the opening of the CP end of the pore. A second cooperative mode shared by all five K(+) channels is the extension of the extracellular and/or CP ends via alternating anticorrelated fluctuations of pairs of diagonally opposite monomers. Residues acting as hinges/anchors in both modes are highly conserved across the members of the family of K(+) channel proteins, consistent with their presently disclosed critical mechanical role in pore gating.

Amino Acid Sequence↗

PubMed2006

Optimal design of protein docking potentials: efficiency and limitations.

Protein-protein docking is a challenging computational problem in functional genomics, particularly when one or both proteins undergo conformational change(s) upon binding. The major challenge is to define scoring function soft enough to tolerate these changes and specific enough to distinguish between near-native and "misdocked" conformations. Using a linear programming technique, we derived protein docking potentials (PDPs) that comply with this requirement. We considered a set of 63 nonredundant complexes to this aim, and generated 400,000 putative docked complexes (decoys) based on shape complementarity criterion for each complex. The PDPs were required to yield for the native (correctly docked) structure a potential energy lower than those of all the nonnative (misdocked) structures. The energy constraints applied to all complexes led to ca. 25 million inequalities, the simultaneous solution of which yielded an optimal set of PDPs that discriminated the correctly docked (up to 4.0 A root-mean-square deviation from known complex structure) structure among the 85 top-ranking (0.02%) decoys in 59/63 examined bound-bound cases. The high performance of the potentials was further verified in jackknife tests and by ranking putative docked conformation submitted to CAPRI. In addition to their utility in identifying correctly folded complexes, the PDPs reveal biologically meaningful features that distinguish docking potentials from folding potentials.

Antigens↗

PubMed2005

Structural changes involved in protein binding correlate with intrinsic motions of proteins in the unbound state.

Protein-protein binding usually involves structural changes that may extend beyond the rearrangements on a local scale, and cannot be explained by a classical lock-and-key mechanism. Several models have been advanced to explain the flexible binding of proteins such as the induced fit mechanism where the ligand is postulated to induce a conformational change at the interaction site upon binding, or the preexisting equilibrium hypothesis that assumes that protein samples an ensemble of conformations at equilibrium conditions and that the ligand binds selectively to an active conformation. We explored the equilibrium motions of proteins that exhibit relatively large (nonlocal) conformational changes upon protein binding using the Gaussian network model and the anisotropic network model of protein dynamics. For four complexes, LIR-1/HLA-A2, Actin/DNase I, CDK2/cyclin, and CDK6/p16(INK4a), the motions calculated for the monomer exhibiting the largest conformational change, in its unbound (free) form, correlate with the experimentally observed structural changes upon binding. This study emphasizes the preexisting equilibrium/conformational selection as a mechanism for protein-protein interaction and lends support the concept that proteins, in their native conformation, are predisposed to undergo conformational fluctuations that are relevant to, or even required for, their biological functions.

Actins↗

PubMed2005

Elastic network models for understanding biomolecular machinery: from enzymes to supramolecular assemblies.

With advances in structure genomics, it is now recognized that knowledge of structure alone is insufficient to understand and control the mechanisms of biomolecular function. Additional information in the form of dynamics is needed. As demonstrated in a large number of studies, the machinery of proteins and their complexes can be understood to a good approximation by adopting Gaussian (or elastic) network models (GNM) for simplified normal mode analyses. While this approximation lacks chemical details, it provides us with a means for assessing the collective motions of large structures/assemblies and perform a comparative analysis of a series of proteins, thus providing insights into the mechanical aspects of biomolecular dynamics. In this paper, we discuss recent applications of GNM to a series of enzymes as well as large structures such as the HK97 bacteriophage viral capsids. Understanding the dynamics of large protein structures can be computationally challenging. To this end, we introduce a new approach for building a hierarchical, reduced rank representation of the protein topology and consequently the fluctuation dynamics.

Bacteriophages↗

PubMed2005

iGNM: a database of protein functional motions based on Gaussian Network Model.

MOTIVATION: The knowledge of protein structure is not sufficient for understanding and controlling its function. Function is a dynamic property. Although protein structural information has been rapidly accumulating in databases, little effort has been invested to date toward systematically characterizing protein dynamics. The recent success of analytical methods based on elastic network models, and in particular the Gaussian Network Model (GNM), permits us to perform a high-throughput analysis of the collective dynamics of proteins. RESULTS: We computed the GNM dynamics for 20 058 structures from the Protein Data Bank, and generated information on the equilibrium dynamics at the level of individual residues. The results are stored on a web-based system called iGNM and configured so as to permit the users to visualize or download the results through a standard web browser using a simple search engine. Static and animated images for describing the conformational mobility of proteins over a broad range of normal modes are accessible, along with an online calculation engine available for newly deposited structures. A case study of the dynamics of 20 non-homologous hydrolases is presented to illustrate the utility of the iGNM database for identifying key residues that control the cooperative motions and revealing the connection between collective dynamics and catalytic activity.

Algorithms↗

PubMed2005

Structural dynamics of nucleosome core particle: comparison with nucleosomes containing histone variants.

The present study provides insights on the dominant mechanisms of motions of the nucleosome core particle and the changes in its functional dynamics in response to histone variants. Comparative analysis of the global dynamics of nucleosomes with native and variant H2A histones, using normal mode analysis revealed that the dynamics of the nucleosome is highly symmetric, and its interaction with the nucleosomal DNA plays a vital role in its regulation. The collective dynamics of nucleosomes are predicted to be dominated by two types of large-scale motions: (1) a global stretching-compression of nucleosome along the dyad axis by which the nucleosome undergoes a breathing motion with a massive distortion of nucleosomal DNA, modulated by histone-DNA interactions; and (2) the flipping (or bending) of both the sides of the nucleosome in an out-of-plane fashion with respect to the dyad axis, originated by the highly dynamic N-termini of H3 and (H2A.Z-H2B) dimer in agreement with the experimentally observed perturbed dynamics of the particular N-terminus under physiological conditions. In general, the nucleosomes with variant histones exhibit higher mobilities and weaker correlations between internal motions compared to the nucleosome containing ordinary histones. The differences are more pronounced at the L1 and L2 loops of the respective monomers H2B and H2A, and at the N-termini of the monomers H3 and H4, all of which closely interact with the wrapping DNA.

Amino Acid Motifs↗

PubMed2005

Coarse-grained normal mode analysis in structural biology.

The realization that experimentally observed functional motions of proteins can be predicted by coarse-grained normal mode analysis has renewed interest in applications to structural biology. Notable applications include the prediction of biologically relevant motions of proteins and supramolecular structures driven by their structure-encoded collective dynamics; the refinement of low-resolution structures, including those determined by cryo-electron microscopy; and the identification of conserved dynamic patterns and mechanically key regions within protein families. Additionally, hybrid methods that couple atomic simulations with deformations derived from coarse-grained normal mode analysis are able to sample collective motions beyond the range of conventional molecular dynamics simulations. Such applications have provided great insight into the underlying principles linking protein structures to their dynamics and their dynamics to their functions.

Binding Sites↗

PubMed2005

Maturation dynamics of bacteriophage HK97 capsid.

Maturation of the bacteriophage HK97 capsid requires a large conformational change of the virus capsid. Experimental studies have identified several intermediates along this maturation pathway. To gain insights into the molecular mechanisms of capsid maturation, we examined the fluctuation dynamics of the procapsid and mature capsid using a residue-level computational approach. The most cooperative motions of the procapsid are found to be consistent with the observed change in configuration that takes place during maturation. A few dominant modes of motion are sufficient to describe the anisotropic expansion that accompanies maturation. Based upon these modes, maturation is proposed to occur via an overall expansion and reconfiguration of the capsid initiated by puckering of the pentamers, followed by flattening and crosslinking of the hexameric subunits, and finally crosslinking of the pentameric subunits. The highly mobile E loops are stabilized by anchoring to highly stable residues belonging to neighboring subunits.

Capsid↗

PubMed2005

Coupling between catalytic site and collective dynamics: a requirement for mechanochemical activity of enzymes.

Growing evidence supports the view that enzymatic activity results from a subtle interplay between chemical kinetics and molecular motions. A systematic analysis is performed here to delineate the type and level of coupling between catalysis and conformational mechanics. The dynamics of a set of 98 enzymes representative of different EC classes are analyzed with the Gaussian network model (GNM) and compared with experimental data. In more than 70% of the examined enzymes, the global hinge centers predicted by the GNM are found to be colocalized with the catalytic sites experimentally identified. Low translational mobility (< 7%) is observed for the catalytic residues, consistent with the fine-tuned design of enzymes to achieve precise mechanochemical activities. Ligand binding sites, while closely neighboring catalytic sites, enjoy a moderate flexibility to accommodate the ligand binding. These findings could serve as additional criteria for assessing drug binding residues and could lessen the computational burden of substrate docking searches.

Binding Sites↗

PubMed2004

A stable three-enzyme creatinine biosensor. 1. Impact of structure, function and environment on PEGylated and immobilized sarcosine oxidase.

The determination of creatinine levels in biological fluids is an increasingly important clinical requirement. Amperometric biosensors have been developed based on a three-enzyme system which converts creatinine to amperometrically measurable hydrogen peroxide. The development of the amperometric creatinine biosensor has been slow due the complexity of the three-enzyme system. This paper, the first of three, discusses the chemical modification of sarcosine oxidase and the immobilization and stabilization of this enzyme using polyurethane prepolymers. Sarcosine oxidase was completely inactivated after modification using poly(ethylene glycol) activated with isocyanate. The addition of a competitive inhibitor during enzyme modification was effective in protecting the enzyme from inactivation. Computational analysis of the structure of sarcosine oxidase suggests that there is a lysine in the active site that may be hyper-reactive. The enzyme was irreversibly immobilized using polyurethane prepolymers and retained significant activity. The enzyme's half-life at 37 degrees C increased from seven days to more than 50 days after immobilization.

Biosensing Techniques↗

PubMed2004

A stable three enzyme creatinine biosensor. 2. Analysis of the impact of silver ions on creatine amidinohydrolase.

The enzyme creatine amidinohydrolase is a clinically important enzyme used in the determination of creatinine in blood and urine. Continuous use biosensors are becoming more important in the clinical setting; however, long-use creatinine biosensors have not been commercialized due to the complexity of the three-enzyme creatinine biosensor and the lack of stability of its components. This paper, the second in a series of three, describes the immobilization and stabilization of creatine amidinohydrolase. Creatine amidinohydrolase modified with poly(ethylene glycol) activated with isocyanate retains significant activity after modification. The enzyme was successfully immobilized into hydrophilic polyurethanes using a reactive prepolymer strategy. The immobilized enzyme retained significant activity over a 30 day period at 37 degrees C and was irreversibly immobilized into the polymer. Despite being stabilized in the polymer, the enzyme remained highly sensitive to silver ions which were released from the amperometric electrodes. Computational analysis of the structure of the protein using the Gaussian network model suggests that the silver ions bind tightly to a cysteine residue preventing normal enzyme dynamics and catalysis.

Biosensing Techniques↗

PubMed2004

Escherichia coli adenylate kinase dynamics: comparison of elastic network model modes with mode-coupling (15)N-NMR relaxation data.

The dynamics of adenylate kinase of Escherichia coli (AKeco) and its complex with the inhibitor AP(5)A, are characterized by correlating the theoretical results obtained with the Gaussian Network Model (GNM) and the anisotropic network model (ANM) with the order parameters and correlation times obtained with Slowly Relaxing Local Structure (SRLS) analysis of (15)N-NMR relaxation data. The AMPbd and LID domains of AKeco execute in solution large amplitude motions associated with the catalytic reaction Mg(+2)*ATP + AMP --> Mg(+2)*ADP + ADP. Two sets of correlation times and order parameters were determined by NMR/SRLS for AKeco, attributed to slow (nanoseconds) motions with correlation time tau( perpendicular) and low order parameters, and fast (picoseconds) motions with correlation time tau( parallel) and high order parameters. The structural connotation of these patterns is examined herein by subjecting AKeco and AKeco*AP(5)A to GNM analysis, which yields the dynamic spectrum in terms of slow and fast modes. The low/high NMR order parameters correlate with the slow/fast modes of the backbone elucidated with GNM. Likewise, tau( parallel) and tau( perpendicular) are associated with fast and slow GNM modes, respectively. Catalysis-related domain motion of AMPbd and LID in AKeco, occurring per NMR with correlation time tau( perpendicular), is associated with the first and second collective slow (global) GNM modes. The ANM-predicted deformations of the unliganded enzyme conform to the functional reconfiguration induced by ligand-binding, indicating the structural disposition (or potential) of the enzyme to bind its substrates. It is shown that NMR/SRLS and GNM/ANM analyses can be advantageously synthesized to provide insights into the molecular mechanisms that control biological function.

Adenylate Kinase↗

PubMed2004

High-efficiency bypass of DNA damage by human DNA polymerase Q.

Endogenous DNA damage arises frequently, particularly apurinic (AP) sites. These must be dealt with by cells in order to avoid genotoxic effects. DNA polymerase theta; is a newly identified enzyme encoded by the human POLQ gene. We find that POLQ has an exceptional ability to bypass an AP site, inserting A with 22% of the efficiency of a normal template, and continuing extension as avidly as with a normally paired base. POLQ preferentially incorporates A opposite an AP site and strongly disfavors C. On nondamaged templates, POLQ makes frequent errors, incorporating G or T opposite T about 1% of the time. This very low fidelity distinguishes POLQ from other A-family polymerases. POLQ has three sequence insertions between conserved motifs in its catalytic site. One insert of approximately 22 residues into the tip of the polymerase thumb subdomain is predicted to confer considerable flexibility and additional DNA contacts to affect enzyme fidelity. POLQ is the only known enzyme that efficiently carries out both the insertion and extension steps for bypass of AP sites, commonly formed as endogenous genomic lesions.

Amino Acid Motifs↗