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1.  On the structure and dynamics of the complex of the nucleosome and the linker histone 
Nucleic Acids Research  2011;39(12):5255-5263.
Several different models of the linker histone (LH)–nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode analysis of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) experiments. GH5 binds asymmetrically with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, respectively, favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.
PMCID: PMC3130272  PMID: 21355036
2.  On Calculation of the Electrostatic Potential of a Phosphatidylinositol Phosphate-Containing Phosphatidylcholine Lipid Membrane Accounting for Membrane Dynamics 
PLoS ONE  2014;9(8):e104778.
Many signaling events require the binding of cytoplasmic proteins to cell membranes by recognition of specific charged lipids, such as phosphoinositol-phosphates. As a model for a protein-membrane binding site, we consider one charged phosphoinositol phosphate (PtdIns(3)P) embedded in a phosphatidylcholine bilayer. As the protein-membrane binding is driven by electrostatic interactions, continuum solvent models require an accurate representation of the electrostatic potential of the phosphoinositol phosphate-containing membrane. We computed and analyzed the electrostatic potentials of snapshots taken at regular intervals from molecular dynamics simulations of the bilayer. We observe considerable variation in the electrostatic potential of the bilayer both along a single simulation and between simulations performed with the GAFF or CHARMM c36 force fields. However, we find that the choice of GAFF or CHARMM c36 parameters has little effect on the electrostatic potential of a given configuration of the bilayer with a PtdIns(3)P embedded in it. From our results, we propose a remedian averaging method for calculating the electrostatic potential of a membrane system that is suitable for simulations of protein-membrane binding with a continuum solvent model.
PMCID: PMC4139306  PMID: 25141217
3.  Long range Debye-Hückel correction for computation of grid-based electrostatic forces between biomacromolecules 
BMC Biophysics  2014;7:4.
Brownian dynamics (BD) simulations can be used to study very large molecular systems, such as models of the intracellular environment, using atomic-detail structures. Such simulations require strategies to contain the computational costs, especially for the computation of interaction forces and energies. A common approach is to compute interaction forces between macromolecules by precomputing their interaction potentials on three-dimensional discretized grids. For long-range interactions, such as electrostatics, grid-based methods are subject to finite size errors. We describe here the implementation of a Debye-Hückel correction to the grid-based electrostatic potential used in the SDA BD simulation software that was applied to simulate solutions of bovine serum albumin and of hen egg white lysozyme.
We found that the inclusion of the long-range electrostatic correction increased the accuracy of both the protein-protein interaction profiles and the protein diffusion coefficients at low ionic strength.
An advantage of this method is the low additional computational cost required to treat long-range electrostatic interactions in large biomacromolecular systems. Moreover, the implementation described here for BD simulations of protein solutions can also be applied in implicit solvent molecular dynamics simulations that make use of gridded interaction potentials.
PMCID: PMC4082500  PMID: 25045516
Continuum solvent electrostatics; Ionic strength; Debye-Hückel; Poisson-Boltzmann equation; Brownian dynamics simulation; Protein diffusion; Discretization grid; Finite difference; Second virial coefficient; Small angle scattering intensity
4.  Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria 
PLoS Computational Biology  2013;9(7):e1003159.
Pyruvate kinase (PYK) is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric activation may reflect adaptation to different environments with different concentrations of activating compounds. The combined computational approach employed can readily be applied to other enzymes.
Author Summary
Some lactic acid bacteria are antibiotic resistant pathogens causing severe diseases whereas others are healthy probiotics used in the food industry. What makes an LAB a friend or a foe and how do they adapt to survive in such different environments? Here, we addressed this problem by focusing on the enzyme pyruvate kinase, which plays a central role in the metabolism of lactic acid bacteria. This enzyme needs to react quickly to changes in the environment and, therefore, its activity is strictly regulated. In this study, we used computational techniques to predict the cellular substances, called allosteric activators that are responsible for the quick and effective activation of pyruvate kinase. We modeled the three dimensional structures of pyruvate kinases from different bacteria and computed interactions with possible activators. We simulated the dynamic behavior of the pyruvate kinase activity and, considering the cellular concentrations of metabolites in the different organisms, predicted the activators. We found that different lactic acid bacteria have different preferences for activators and that the level of activator specificity can be related to the environment in which the bacteria live.
PMCID: PMC3738050  PMID: 23946717
5.  Targeting protein dynamics in drug design 
Journal of Cheminformatics  2013;5(Suppl 1):O1.
PMCID: PMC3606222
6.  Translational repression of thymidylate synthase by targeting its mRNA 
Nucleic Acids Research  2013;41(7):4159-4170.
Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA.
PMCID: PMC3627590  PMID: 23423353
7.  The Interaction Properties of the Human Rab GTPase Family – A Comparative Analysis Reveals Determinants of Molecular Binding Selectivity 
PLoS ONE  2012;7(4):e34870.
Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.
Methodology/Principal Findings
Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics.
We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity.
PMCID: PMC3327705  PMID: 22523562
8.  Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9 
PLoS Computational Biology  2011;7(8):e1002152.
The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.
Author Summary
We describe the first atomic-detail models and simulations of a full-length, membrane-bound mammalian cytochrome P450. To date, all the structural studies of microsomal, drug-metabolizing cytochrome P450s have been performed using engineered, solubilized forms of the enzymes and it is not yet understood how the membrane influences their structure, dynamics, and ability to bind substrates. We focused on CYP2C9, the second most abundant cytochrome P450 in the human liver which oxidizes 20% of all marketed drugs. Here, we have derived models of CYP2C9-membrane complexes with a modeling procedure based on molecular dynamics simulations started with arbitrary configurations of the protein in the membrane and performed using both coarse grained and atomic-detail molecular representations. This procedure is expected to be generally applicable to proteins that are anchored in the membrane with a single transmembrane helix. The models and simulations provide evidence for selective substrate access and product release routes in membrane-bound CYPs. This observation may contribute to new strategies to manipulate the activity of CYPs and other enzymes with buried active sites. We anticipate that this study will bring about a paradigm shift towards studying microsomal CYPs as dynamic structures in their natural, lipid environment rather than in artificially solubilized forms.
PMCID: PMC3154944  PMID: 21852944
9.  Diffusion of hydrophobin proteins in solution and interactions with a graphite surface 
BMC Biophysics  2011;4:9.
Hydrophobins are small proteins produced by filamentous fungi that have a variety of biological functions including coating of spores and surface adhesion. To accomplish these functions, they rely on unique interface-binding properties. Using atomic-detail implicit solvent rigid-body Brownian dynamics simulations, we studied the diffusion of HFBI, a class II hydrophobin from Trichoderma reesei, in aqueous solution in the presence and absence of a graphite surface.
In the simulations, HFBI exists in solution as a mixture of monomers in equilibrium with different types of oligomers. The oligomerization state depends on the conformation of HFBI. When a Highly Ordered Pyrolytic Graphite (HOPG) layer is present in the simulated system, HFBI tends to interact with the HOPG layer through a hydrophobic patch on the protein.
From the simulations of HFBI solutions, we identify a tetrameric encounter complex stabilized by non-polar interactions between the aliphatic residues in the hydrophobic patch on HFBI. After the formation of the encounter complex, a local structural rearrangement at the protein interfaces is required to obtain the tetrameric arrangement seen in HFBI crystals. Simulations performed with the graphite surface show that, due to a combination of a geometric hindrance and the interaction of the aliphatic sidechains with the graphite layer, HFBI proteins tend to accumulate close to the hydrophobic surface.
PMCID: PMC3114038  PMID: 21595866
10.  Diffusion and association processes in biological systems: theory, computation and experiment 
BMC Biophysics  2011;4:2.
Macromolecular diffusion plays a fundamental role in biological processes. Here, we give an overview of recent methodological advances and some of the challenges for understanding how molecular diffusional properties influence biological function that were highlighted at a recent workshop, BDBDB2, the second Biological Diffusion and Brownian Dynamics Brainstorm.
PMCID: PMC3093674  PMID: 21595997
11.  webPIPSA: a web server for the comparison of protein interaction properties 
Nucleic Acids Research  2008;36(Web Server issue):W276-W280.
Protein molecular interaction fields are key determinants of protein functionality. PIPSA (Protein Interaction Property Similarity Analysis) is a procedure to compare and analyze protein molecular interaction fields, such as the electrostatic potential. PIPSA may assist in protein functional assignment, classification of proteins, the comparison of binding properties and the estimation of enzyme kinetic parameters. webPIPSA is a web server that enables the use of PIPSA to compare and analyze protein electrostatic potentials. While PIPSA can be run with downloadable software (see, webPIPSA extends and simplifies a PIPSA run. This allows non-expert users to perform PIPSA for their protein datasets. With input protein coordinates, the superposition of protein structures, as well as the computation and analysis of electrostatic potentials, is automated. The results are provided as electrostatic similarity matrices from an all-pairwise comparison of the proteins which can be subjected to clustering and visualized as epograms (tree-like diagrams showing electrostatic potential differences) or heat maps. webPIPSA is freely available at:
PMCID: PMC2447742  PMID: 18420653
12.  qPIPSA: Relating enzymatic kinetic parameters and interaction fields 
BMC Bioinformatics  2007;8:373.
The simulation of metabolic networks in quantitative systems biology requires the assignment of enzymatic kinetic parameters. Experimentally determined values are often not available and therefore computational methods to estimate these parameters are needed. It is possible to use the three-dimensional structure of an enzyme to perform simulations of a reaction and derive kinetic parameters. However, this is computationally demanding and requires detailed knowledge of the enzyme mechanism. We have therefore sought to develop a general, simple and computationally efficient procedure to relate protein structural information to enzymatic kinetic parameters that allows consistency between the kinetic and structural information to be checked and estimation of kinetic constants for structurally and mechanistically similar enzymes.
We describe qPIPSA: quantitative Protein Interaction Property Similarity Analysis. In this analysis, molecular interaction fields, for example, electrostatic potentials, are computed from the enzyme structures. Differences in molecular interaction fields between enzymes are then related to the ratios of their kinetic parameters. This procedure can be used to estimate unknown kinetic parameters when enzyme structural information is available and kinetic parameters have been measured for related enzymes or were obtained under different conditions. The detailed interaction of the enzyme with substrate or cofactors is not modeled and is assumed to be similar for all the proteins compared. The protein structure modeling protocol employed ensures that differences between models reflect genuine differences between the protein sequences, rather than random fluctuations in protein structure.
Provided that the experimental conditions and the protein structural models refer to the same protein state or conformation, correlations between interaction fields and kinetic parameters can be established for sets of related enzymes. Outliers may arise due to variation in the importance of different contributions to the kinetic parameters, such as protein stability and conformational changes. The qPIPSA approach can assist in the validation as well as estimation of kinetic parameters, and provide insights into enzyme mechanism.
PMCID: PMC2174957  PMID: 17919319
13.  MolSurfer: a macromolecular interface navigator 
Nucleic Acids Research  2003;31(13):3349-3351.
We describe the current status of the Java molecular graphics tool, MolSurfer. MolSurfer has been designed to assist the analysis of the structures and physico-chemical properties of macromolecular interfaces. MolSurfer provides a coupled display of two-dimensional (2D) maps of the interfaces generated with the ADS software and a three-dimensional (3D) view of the macromolecular structure in the Java PDB viewer, WebMol. The interfaces are analytically defined and properties such as electrostatic potential or hydrophobicity are projected on to them. MolSurfer has been applied previously to analyze a set of 39 protein–protein complexes, with structures available from the Protein Data Bank (PDB). A new application, described here, is the visualization of 75 interfaces in structures of protein–DNA and protein–RNA complexes. Another new feature is that the MolSurfer web server is now able to compute and map Poisson–Boltzmann electrostatic potentials of macromolecules onto interfaces. The MolSurfer web server is available at
PMCID: PMC168994  PMID: 12824324
14.  DSMM: a Database of Simulated Molecular Motions 
Nucleic Acids Research  2003;31(1):456-457.
We describe a Database of Simulated Molecular Motions (DSMM). This database is designed to serve as a single searchable site for locating movies and animations from simulations of biomolecules. DSMM is accessible via a webserver at:
PMCID: PMC165560  PMID: 12520051

Results 1-14 (14)