Generation of native-like protein structures from limited NMR data, modern force fields and advanced conformational sampling.

Determining an accurate initial native-like protein fold is one of the most important and time-consuming steps of de novo NMR structure determination. Here we demonstrate that high-quality native-like models can be rapidly generated from initial structures obtained using limited NOE assignments, through replica exchange molecular dynamics refinement with a generalized Born implicit solvent (REX/GB). Conventional structure calculations using an initial sparse NOE set were unable to identify a unique topology for the zinc-bound C-terminal domain of E.

A nonadditive methanol force field: bulk liquid and liquid-vapor interfacial properties via molecular dynamics simulations using a fluctuating charge model.

We study the bulk and interfacial properties of methanol via molecular dynamics simulations using a CHARMM (Chemistry at HARvard Molecular Mechanics) fluctuating charge force field. We discuss the parametrization of the electrostatic model as part of the ongoing CHARMM development for polarizable protein force fields. The bulk liquid properties are in agreement with available experimental data and competitive with existing fixed-charge and polarizable force fields.

The requirement for mechanical coupling between head and S2 domains in smooth muscle myosin ATPase regulation and its implications for dimeric motor function.

A combination of experimental structural data, homology modelling and elastic network normal mode analysis is used to explore how coupled motions between the two myosin heads and the dimerization domain (S2) in smooth muscle myosin II determine the domain movements required to achieve the inhibited state of this ATP-dependent molecular motor. These physical models rationalize the empirical requirement for at least two heptads of non-coiled alpha-helix at the junction between the myosin heads and S2, and the dependence of regulation on S2 length. The results correlate well with biochemical data regarding altered conformational-dependent solubility and stability.

Refinement of NMR structures using implicit solvent and advanced sampling techniques.

NMR biomolecular structure calculations exploit simulated annealing methods for conformational sampling and require a relatively high level of redundancy in the experimental restraints to determine quality three-dimensional structures. Recent advances in generalized Born (GB) implicit solvent models should make it possible to combine information from both experimental measurements and accurate empirical force fields to improve the quality of NMR-derived structures. In this paper, we study the influence of implicit solvent on the refinement of protein NMR structures and identify an optimal protocol of utilizing these improved force fields.

Receptor rigidity and ligand mobility in trypsin-ligand complexes.

The trypsin-like serine proteases comprise a structurally similar family of proteins with a wide diversity of biological functions. Members of this family play roles in digestion, hemostasis, immune responses, and cancer metastasis. Bovine trypsin is an archetypical member of this family that has been extensively characterized both functionally and structurally, and that preferentially hydrolyzes Arg/Lys-Xaa peptide bonds.

Diversity and identity of mechanical properties of icosahedral viral capsids studied with elastic network normal mode analysis.

We analyze the mechanical properties and putative dynamical fluctuations of a variety of viral capsids comprising different sizes and quasi-equivalent symmetries by performing normal mode analysis using the elastic network model. The expansion of the capsid to a swollen state is studied using normal modes and is compared with the experimentally observed conformational change for three of the viruses for which experimental data exist. We show that a combination of one or two normal modes captures remarkably well the overall translation that dominates the motion between the two conformational states, and reproduces the overall conformational change.

A modified TIP3P water potential for simulation with Ewald summation.

The charges and Lennard-Jones parameters of the TIP3P water potential have been modified to improve its performance under the common condition for molecular dynamics simulations of using Ewald summation in lieu of relatively short nonbonded truncation schemes. These parameters were optimized under the condition that the hydrogen atoms do not have Lennard-Jones parameters, thus making the model independent of the combining rules used for the calculation of nonbonded, heteroatomic interaction energies, and limiting the number of Lennard-Jones calculations required. Under these conditions, this model provides accurate density (rho = 0.

Mechanism for ordered receptor binding by human prolactin.

Prolactin, a lactogenic hormone, binds to two prolactin receptors sequentially, the first receptor binding at site 1 of the hormone followed by the second receptor binding at site 2. We have investigated the mechanism by which human prolactin (hPRL) binds the extracellular domain of the human prolactin receptor (hPRLbp) using surface plasmon resonance (SPR) technology. We have covalently coupled hPRL to the SPR chip surface via coupling chemistries that reside in and block either site 1 or site 2.

Normal mode based flexible fitting of high-resolution structure into low-resolution experimental data from cryo-EM.

A new method for the flexible fitting of high-resolution structures into low-resolution maps of macromolecular complexes from electron microscopy has been recently described in applications to simulated electron density maps. This method uses a linear combination of low-frequency normal modes in an iterative manner to deform the structure optimally to conform to the low-resolution electron density map. Gradient-following techniques in the coordinate space of collective normal modes are used to optimize the overall correlation coefficient between computed and measured electron densities.

The coupling of structural fluctuations to hydride transfer in dihydrofolate reductase.

The energy barrier for hydride transfer in wild-type G121V and G121S variants of Escherichia coli dihydrofolate reductase (DHFR) fluctuates in a time-dependent manner. This fluctuation may be attributed to structural changes in the protein that modulate the site of chemistry. Despite being far from the active site, mutations at position 121 of DHFR reduce the hydride transfer rate of the enzyme.

An electrostatic basis for the stability of thermophilic proteins.

Two factors provide key contributions to the stability of thermophilic proteins relative to their mesophilic homologues: electrostatic interactions of charged residues in the folded state and the dielectric response of the folded protein. The dielectric response for proteins in a "thermophilic series" globally modulates the thermal stability of its members, with the calculated dielectric constant for the protein increasing from mesophiles to hyperthermophiles. This variability results from differences in the distribution of charged residues on the surface of the protein, in agreement with structural and genetic observations.

Constant-pH molecular dynamics using continuous titration coordinates.

In this work, we explore the question of whether pK(a) calculations based on a microscopic description of the protein and a macroscopic description of the solvent can be implemented to examine conformationally dependent proton shifts in proteins. To this end, we introduce a new method for performing constant-pH molecular dynamics (PHMD) simulations utilizing the generalized Born implicit solvent model. This approach employs an extended Hamiltonian in which continuous titration coordinates propagate simultaneously with the atomic motions of the system.

Ribosome motions modulate electrostatic properties.

The electrostatic properties of the 70S ribosome of Thermus thermophilus were studied qualitatively by solving the Poisson-Boltzmann (PB) equation in aqueous solution and with physiological ionic strength. The electrostatic potential was calculated for conformations of the ribosome derived by recent normal mode analysis (Tama, F., et al.

Implicit solvation based on generalized Born theory in different dielectric environments.

In this paper we are investigating the effect of the dielectric environment on atomic Born radii used in generalized Born (GB) methods. Motivated by the Kirkwood expression for the reaction field of a single off-center charge in a spherical cavity, we are proposing extended formalisms for the calculation of Born radii as a function of external and internal dielectric constants. We demonstrate that reaction field energies calculated from environmentally dependent Born radii lead to much improved agreement with Poisson-Boltzmann solutions for low dielectric external environments, such as biological membranes or organic solvent, compared to previous methods where the calculation of Born radii does not depend on the environment.

CHARMM fluctuating charge force field for proteins: II protein/solvent properties from molecular dynamics simulations using a nonadditive electrostatic model.

A fluctuating charge (FQ) force field is applied to molecular dynamics simulations for six small proteins in explicit polarizable solvent represented by the TIP4P-FQ potential. The proteins include 1FSV, 1ENH, 1PGB, 1VII, 1H8K, and 1CRN, representing both helical and beta-sheet secondary structural elements. Constant pressure and temperature (NPT) molecular dynamics simulations are performed on time scales of several nanoseconds, the longest simulations yet reported using explicitly polarizable all-atom empirical potentials (for both solvent and protein) in the condensed phase.

Model-free analysis of protein dynamics: assessment of accuracy and model selection protocols based on molecular dynamics simulation.

The popular model-free approach to analyze NMR relaxation measurements has been examined using artificial amide (15)N relaxation data sets generated from a 10 nanosecond molecular dynamics trajectory of a dihydrofolate reductase ternary complex in explicit water. With access to a detailed picture of the underlying internal motions, the efficacy of model-free analysis and impact of model selection protocols on the interpretation of NMR data can be studied. In the limit of uncorrelated global tumbling and internal motions, fitting the relaxation data to the model-free models can recover a significant amount of quantitative information on the internal dynamics.

Gatekeepers in the ribosomal protein s6: thermodynamics, kinetics, and folding pathways revealed by a minimalist protein model.

We investigate the effect of structural gatekeepers on the folding of the ribosomal protein S6. Folding thermodynamics and early refolding kinetics are studied for this system utilizing computer simulations of a minimalist protein model. When gatekeepers are eliminated, the thermodynamic signature of a folding intermediate emerges, and a marked decrease in folding efficiency is observed.

Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations.

Computational studies of proteins based on empirical force fields represent a powerful tool to obtain structure-function relationships at an atomic level, and are central in current efforts to solve the protein folding problem. The results from studies applying these tools are, however, dependent on the quality of the force fields used. In particular, accurate treatment of the peptide backbone is crucial to achieve representative conformational distributions in simulation studies.

Assessing scoring functions for protein-ligand interactions.

An assessment of nine scoring functions commonly applied in docking using a set of 189 protein-ligand complexes is presented. The scoring functions include the CHARMm potential, the scoring function DrugScore, the scoring function used in AutoDock, the three scoring functions implemented in DOCK, as well as three scoring functions implemented in the CScore module in SYBYL (PMF, Gold, ChemScore). We evaluated the abilities of these scoring functions to recognize near-native configurations among a set of decoys and to rank binding affinities.

MMTSB Tool Set: enhanced sampling and multiscale modeling methods for applications in structural biology.

We describe the Multiscale Modeling Tools for Structural Biology (MMTSB) Tool Set (https://mmtsb.scripps.edu/software/mmtsbToolSet.

Recent advances in the development and application of implicit solvent models in biomolecule simulations.

Advances have recently been made in the development of implicit solvent methodologies and their application to the modeling of biomolecules, particularly with regard to generalized Born approaches, dielectric screening function formulations and models based on solvent-accessible surface areas. Interesting new developments include more refined non-polar solvation energy estimators, and implicit methods for modeling low-dielectric and heterogeneous environments such as membrane systems. These have been successfully applied to molecular dynamics simulations, the scoring of protein conformations, and the calculation of binding affinities and folding free energy landscapes.

Comparative study of several algorithms for flexible ligand docking.

We have performed a comparative assessment of several programs for flexible molecular docking: DOCK 4.0, FlexX 1.8, AutoDock 3.

Efficient approximate all-atom solvent accessible surface area method parameterized for folded and denatured protein conformations.

Continuing advances in computer hardware and software are permitting atomic-resolution molecular simulations for longer time scales and on larger systems. Despite these advances, routinely performing atomistic simulations with explicit water for even small proteins, which reach the folding time of such proteins, remains intractable for the foreseeable future. An implicit approximation of the solvent environment using a solvent accessible surface area (SASA) term in a molecular mechanics potential function allows exclusion of the explicit water molecules in protein simulations.

Flexible multi-scale fitting of atomic structures into low-resolution electron density maps with elastic network normal mode analysis.

A novel method is presented for the quantitative flexible docking of high-resolution structure into low-resolution maps of macromolecular complexes from electron microscopy. This method uses a linear combination of low-frequency normal modes from elastic network description of the molecular framework in an iterative manner to deform the structure optimally to conform to the low-resolution electron density map. The methodology utilizes gradient following techniques in collective normal modes to locally optimize the overall correlation coefficient between computed and measured electron density.

De novo folding of membrane proteins: an exploration of the structure and NMR properties of the fd coat protein.

De novo folding simulations of the major pVIII coat protein from filamentous fd bacteriophage, using a newly developed implicit membrane generalized Born model and replica-exchange molecular dynamics, are presented and discussed. The quality of the predicted structures, judged by comparison of the root-mean-square deviations of a room temperature ensemble of conformations from the replica-exchange simulations and experimental structures from both solid-state NMR in lipid bilayers and solution-phase NMR on the protein in micelles, was quite good, reinforcing the general quality of the folding simulations. The transmembrane helical segment of the protein was well defined in comparison with experiment and the amphipathic helical fragment remained at the membrane/aqueous phase boundary while undergoing significant conformational flexibility due to the loop connecting the two helical segments of the protein.