Biomedical subjects

Charles L Brooks

Publications and source records attributed to Charles L Brooks.

At least 19 recordsLinked to original sources

PubMed2006

Exploring atomistic details of pH-dependent peptide folding.

Modeling pH-coupled conformational dynamics allows one to probe many important pH-dependent biological processes, ranging from ATP synthesis, enzyme catalysis, and membrane fusion to protein folding/misfolding and amyloid formation. This work illustrates the strengths and capabilities of continuous constant pH molecular dynamics in exploring pH-dependent conformational transitions in proteins by revisiting an experimentally well studied model protein fragment, the C peptide from ribonuclease A. The simulation data reveal a bell-shaped pH profile for the total helix content, in agreement with experiment, and several pairs of electrostatic interactions that control the relative populations of unfolded and partially folded states of various helical lengths. The latter information greatly complements and extends that attainable by current experimental techniques. The present work paves the way for new and exciting applications, such as the study of pH-dependent molecular mechanism in the formation of amyloid comprising peptides from Alzheimer's and Parkinson's diseases.

Amino Acid Substitution↗

PubMed2006

Harmonic Fourier beads method for studying rare events on rugged energy surfaces.

We present a robust, distributable method for computing minimum free energy paths of large molecular systems with rugged energy landscapes. The method, which we call harmonic Fourier beads (HFB), exploits the Fourier representation of a path in an appropriate coordinate space and proceeds iteratively by evolving a discrete set of harmonically restrained path points-beads-to generate positions for the next path. The HFB method does not require explicit knowledge of the free energy to locate the path. To compute the free energy profile along the final path we employ an umbrella sampling method in two generalized dimensions. The proposed HFB method is anticipated to aid the study of rare events in biomolecular systems. Its utility is demonstrated with an application to conformational isomerization of the alanine dipeptide in gas phase.

Alanine↗

PubMed2006

Membrane assembly of simple helix homo-oligomers studied via molecular dynamics simulations.

The assembly of simple transmembrane helix homo-oligomers is studied by combining a generalized Born implicit membrane model with replica exchange molecular dynamics simulations to sample the conformational space of various oligomerization states and the native oligomeric conformation. Our approach is applied to predict the structures of transmembrane helices of three proteins--glycophorin A, the M2 proton channel, and phospholamban--using only peptide sequence and the native oligomerization state information. In every case, the methodology reproduces native conformations that are in good agreement with available experimental structural data. Thus, our method should be useful in the prediction of native structures of transmembrane domains of other peptides. When we ignore the experimental constraint on the native oligomerization state and attempt de novo prediction of the structure and oligomerization state based only on sequence and simple energetic considerations, we identify the pentamer as the most stable oligomer for phospholamban. However, for the glycophorin A and the M2 proton channels, we tend to predict higher oligomers as more stable. Our studies demonstrate that reliable predictions of the structure of transmembrane helical oligomers can be achieved when the observed oligomerization state is imposed as a constraint, but that further efforts are needed for the de novo prediction of both structure and oligomeric state.

Cell Membrane↗

PubMed2006

Antibody evolution constrains conformational heterogeneity by tailoring protein dynamics.

The evolution of proteins with novel function is thought to start from precursor proteins that are conformationally heterogeneous. The corresponding genes may be duplicated and then mutated to select and optimize a specific conformation. However, testing this idea has been difficult because of the challenge of quantifying protein flexibility and conformational heterogeneity as a function of evolution. Here, we report the characterization of protein heterogeneity and dynamics as a function of evolution for the antifluorescein antibody 4-4-20. Using nonlinear laser spectroscopy, surface plasmon resonance, and molecular dynamics simulations, we demonstrate that evolution localized the Ab-combining site from a heterogeneous ensemble of conformations to a single conformation by introducing mutations that act cooperatively and over significant distances to rigidify the protein. This study demonstrates how protein dynamics may be tailored by evolution and has important implications for our understanding of how novel protein functions are evolved.

Animals↗

PubMed2006

Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release.

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 A by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 +/- 30 Ca2+ ions bound to the capsid and 420 +/- 25 Mg2+ ions thought to be in the interior of the capsid. Depletion of both Ca2+ and Mg2+ ions from RCNMV leads to significant structural changes, including (i) formation of 11- to 13-A-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca2+ and Mg2+ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.

Amino Acid Sequence↗

PubMed2006

Toward the accurate first-principles prediction of ionization equilibria in proteins.

The calculation of pK(a) values for ionizable sites in proteins has been traditionally based on numerical solutions of the Poisson-Boltzmann equation carried out using a high-resolution protein structure. In this paper, we present a method based on continuous constant pH molecular dynamics (CPHMD) simulations, which allows the first-principles description of protein ionization equilibria. Our method utilizes an improved generalized Born implicit solvent model with an approximate Debye-Hückel screening function to account for salt effects and the replica-exchange (REX) protocol for enhanced conformational and protonation state sampling. The accuracy and robustness of the present method are demonstrated by 1 ns REX-CPHMD titration simulations of 10 proteins, which exhibit anomalously large pK(a) shifts for the carboxylate and histidine side chains. The experimental pK(a) values of these proteins are reliably reproduced with a root-mean-square error ranging from 0.6 unit for proteins containing few buried ionizable side chains to 1.0 unit or slightly higher for proteins containing ionizable side chains deeply buried in the core and experiencing strong charge-charge interactions. This unprecedented level of agreement with experimental benchmarks for the de novo calculation of pK(a) values suggests that the CPHMD method is maturing into a practical tool for the quantitative prediction of protein ionization equilibria, and this, in turn, opens a door to atomistic simulations of a wide variety of pH-coupled conformational phenomena in biological macromolecules such as protein folding or misfolding, aggregation, ligand binding, membrane interaction, and catalysis.

Hydrogen-Ion Concentration↗

PubMed2006

Electrostatic properties of cowpea chlorotic mottle virus and cucumber mosaic virus capsids.

Electrostatic properties of cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CMV) were investigated using numerical solutions to the Poisson-Boltzmann equation. Experimentally, it has been shown that CCMV particles swell in the absence of divalent cations when the pH is raised from 5 to 7. CMV, although structurally homologous, does not undergo this transition. An analysis of the calculated electrostatic potential confirms that a strong electrostatic repulsion at the calcium-binding sites in the CCMV capsid is most likely the driving force for the capsid swelling process during the release of calcium. The binding interaction between the encapsulated genome material (RNA) inside of the capsid and the inner capsid shell is weakened during the swelling transition. This probably aids in the RNA release process, but it is unlikely that the RNA is released through capsid openings due to unfavorable electrostatic interaction between the RNA and capsid inner shell residues at these openings. Calculations of the calcium binding energies show that Ca(2+) can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca(2+) ions can induce the capsid contraction and stabilize the native form.

Binding Sites↗

PubMed2006

Revisiting the hexane-water interface via molecular dynamics simulations using nonadditive alkane-water potentials.

We present results addressing properties of a polarizable force field for hexane based on the fluctuating charge (FQ) formalism and developed in conjunction with the Chemistry at Harvard Molecular Mechanics (CHARMM) potential function. Properties of bulk neat hexane, its liquid-vapor interface, and its interface with a polarizable water model (TIP4P-FQ) are discussed. The FQ model is compared to a recently modified alkane model, C27r, also based on the CHARMM potential energy function. With respect to bulk properties, both models predict bulk density within 1%; the FQ model predicts the liquid vaporization enthalpy within 2%, while the C27r force field underestimates the property by roughly 20% (and in this sense reflects the quality of the C27r force field across the spectrum of linear and branched alkanes). The FQ hexane model realistically captures the dielectric properties of the bulk in terms of a dielectric constant of 1.94, in excellent agreement with experimental values in the range of 1.9-2.02. This behavior is also in conformity with a recent polarizable alkane model based on Drude oscillators. Furthermore, the bulk dielectric is essentially captured in the infinite frequency, or optical, dielectric contribution. The FQ model is in this respect a more realistic force field for modeling lipid bilayer interiors for which most current state-of-the-art force fields do not accurately capture the dielectric environment. The molecular polarizability of the FQ model is 11.79 A3, in good agreement with the range of experimental and ab initio values. In contrast to FQ models of polar solvents such as alcohols and water, there was no need to scale gas-phase polarizabilities in order to avoid polarization catastrophes in the pure bulk. In terms of the liquid-vapor and liquid-liquid interfaces, the FQ model displays a rich orientational structure of alkane and water in the respective interfacial systems, in general conforming with earlier simulation studies of such interfaces. The FQ force field shows a marked deviation in the interfacial dipole potentials computed from the charge densities averaged over simulation trajectories. At the liquid-vapor interface, the FQ model predicts a potential drop of -178.71 mV in contrast to the C27r estimate of -433.80 mV. For the hexane-water interface, the FQ force field predicts a dipole potential drop of -379.40 mV in contrast to the C27r value of -105.42 mV. Although the surface dipole potential predicted by the FQ model is roughly 3.5 times that predicted by the C27r potential, it is consistent with reported experimental potentials across solvated lipid bilayers in the range of 400-600 mV.

Alkanes↗

PubMed2006

A line integral reaction path approximation for large systems via nonlinear constrained optimization: application to alanine dipeptide and the beta hairpin of protein G.

A variation of the line integral method of Elber with self-avoiding walk has been implemented using a state of the art nonlinear constrained optimization procedure. The new implementation appears to be robust in finding approximate reaction paths for small and large systems. Exact transition states and intermediates for the resulting paths can easily be pinpointed with subsequent application of the conjugate peak refinement method [S. Fischer and M. Karplus, Chem. Phys. Lett. 194, 252 (1992)] and unconstrained minimization, respectively. Unlike previous implementations utilizing a penalty function approach, the present implementation generates an exact solution of the underlying problem. Most importantly, this formulation does not require an initial guess for the path, which makes it particularly useful for studying complex molecular rearrangements. The method has been applied to conformational rearrangements of the alanine dipeptide in the gas phase and in water, and folding of the beta hairpin of protein G in water. In the latter case a procedure was developed to systematically sample the potential energy surface underlying folding and reconstruct folding pathways within the nearest-neighbor hopping approximation.

Alanine↗

PubMed2006

Albumin binding to FcRn: distinct from the FcRn-IgG interaction.

The MHC-related Fc receptor for IgG (FcRn) protects albumin and IgG from degradation by binding both proteins with high affinity at low pH in the acid endosome and diverting both from a lysosomal pathway, returning them to the extracellular compartment. Immunoblotting and surface plasmon resonance studies show that both IgG and albumin bind noncooperatively to distinct sites on FcRn, that the affinity of FcRn for albumin decreases approximately 200-fold from acidic to neutral pH, and that the FcRn-albumin interaction shows rapid association and dissociation kinetics. Isothermal titration calorimetry shows that albumin binds FcRn with a 1:1 stoichiometry and the interaction has hydrophobic features as evidenced by a large positive change in entropy upon binding. Our results suggest that the FcRn-albumin interaction has unique features distinct from FcRn-IgG binding despite the overall similarity in the pH-dependent binding mechanism by which both ligands are protected from degradation.

Animals↗

PubMed2006

Coiled coils at the edge of configurational heterogeneity. Structural analyses of parallel and antiparallel homotetrameric coiled coils reveal configurational sensitivity to a single solvent-exposed amino acid substitution.

A detailed understanding of the mechanisms by which particular amino acid sequences can give rise to more than one folded structure, such as for proteins that undergo large conformational changes or misfolding, is a long-standing objective of protein chemistry. Here, we describe the crystal structures of a single coiled-coil peptide in distinct parallel and antiparallel tetrameric configurations and further describe the parallel or antiparallel crystal structures of several related peptide sequences; the antiparallel tetrameric assemblies represent the first crystal structures of GCN4-derived peptides exhibiting such a configuration. Intriguingly, substitution of a single solvent-exposed residue enabled the parallel coiled-coil tetramer GCN4-pLI to populate the antiparallel configuration, suggesting that the two configurations are close enough in energy for subtle sequence changes to have important structural consequences. We present a structural analysis of the small changes to the helix register and side-chain conformations that accommodate the two configurations and have supplemented these results using solution studies and a molecular dynamics energetic analysis using a replica exchange methodology. Considering the previous examples of structural nonspecificity in coiled-coil peptides, the findings reported here not only emphasize the predisposition of the coiled-coil motif to adopt multiple configurations but also call attention to the associated risk that observed crytstal structures may not represent the only (or even the major) species present in solution.

Amino Acid Sequence↗

PubMed2006

Balancing solvation and intramolecular interactions: toward a consistent generalized Born force field.

The efficient and accurate characterization of solvent effects is a key element in the theoretical and computational study of biological problems. Implicit solvent models, particularly generalized Born (GB) continuum electrostatics, have emerged as an attractive tool to study the structure and dynamics of biomolecules in various environments. Despite recent advances in this methodology, there remain limitations in the parametrization of many of these models. In the present work, we demonstrate that it is possible to achieve a balanced implicit solvent force field by further optimizing the input atomic radii in combination with adjusting the protein backbone torsional energetics. This parameter optimization is guided by the potentials of mean force (PMFs) between amino acid polar groups, calculated from explicit solvent free energy simulations, and by conformational equilibria of short peptides, obtained from extensive folding and unfolding replica exchange molecular dynamics (REX-MD) simulations. Through the application of this protocol, the delicate balance between the competing solvation forces and intramolecular forces appears to be better captured, and correct conformational equilibria for a range of both helical and beta-hairpin peptides are obtained. The same optimized force field also successfully folds both beta-hairpin trpzip2 and mini-protein Trp-Cage, indicating that it is quite robust. Such a balanced, physics-based force field will be highly applicable to a range of biological problems including protein folding and protein structural dynamics.

Amino Acid Sequence↗

PubMed2006

VIPERdb: a relational database for structural virology.

VIPERdb (http://viperdb.scripps.edu) is a database for icosahedral virus capsid structures. Our aim is to provide a comprehensive resource specific to the needs of the structural virology community, with an emphasis on the description and comparison of derived data from structural and energetic analyses of capsids. A relational database implementation based on a schema for macromolecular structure makes the data highly accessible to the user, allowing detailed queries at the atomic level. Together with curation practices that maintain data uniformity, this will facilitate structural bioinformatics studies of virus capsids. User friendly search, visualization and educational tools on the website allow both structural and derived data to be examined easily and extensively. Links to relevant literature, sequence and taxonomy databases are provided for each entry.

Capsid↗

PubMed2006

Model of the toxic complex of anthrax: responsive conformational changes in both the lethal factor and the protective antigen heptamer.

The toxic complex of anthrax is formed when the monomeric protective antigen (PA) (83 kDa), while bound to its cell-surface receptor, is first converted to PA63 heptamers (PA63h) following N-terminal proteolytic cleavage, and then lethal (LF) (90 kDa) or edema factor (EF) binds to the heptamer. We report a "pseudoatomic" model for the complex of PA63h and full-length LF determined by applying the normal-mode flexible fitting procedure to a approximately 18 A cryo-electron microscopy (EM) density map of the complex. The model describes the interacting surface that buries a total area of approximately 10,140 A2 comprising approximately 40% charged, and approximately 30% each of polar and hydrophobic residues. For the heptamer, the buried surface, composed of approximately 110 residues, involves primarily three monomers and includes for two, similar stretches of the polypeptide chain from domain 1. For LF, the interface again involves approximately 110 residues, mostly from the N-terminal domain I (LF(N)), and the structurally homologous C-terminal domain IV. Most interestingly, bound LF displays a marked conformational change resulting from a "collapse" of domains I, III, and IV on domain II, with the largest movement of approximately 9 A noted for domain I. On the other hand, primarily, rigid-body movements, larger than approximately 10 A for three PA63 monomers, cause the hourglass-shaped heptamer lumen to enlarge by as much as approximately 50% near the middle of the molecule. Such concerted structural rearrangements in LF and the heptamer can facilitate ingress of the ligand into the heptamer lumen prior to unfolding and release through the PA63h channel formed in the acidic late endosomal membrane.

Antigens, Bacterial↗

PubMed2006

Symmetry, form, and shape: guiding principles for robustness in macromolecular machines.

Computational studies of large macromolecular assemblages have come a long way during the past 10 years. With the explosion of computer power and parallel computing, timescales of molecular dynamics simulations have been extended far beyond the hundreds of picoseconds timescale. However, limitations remain for studies of large-scale conformational changes occurring on timescales beyond nanoseconds, especially for large macromolecules. In this review, we describe recent methods based on normal mode analysis that have enabled us to study dynamics on the microsecond timescale for large macromolecules using different levels of coarse graining, from atomically detailed models to those employing only low-resolution structural information. Emerging from such studies is a control principle for robustness in Nature's machines. We discuss this idea in the context of large-scale functional reorganization of the ribosome, virus particles, and the muscle protein myosin.

Algorithms↗

PubMed2005

Application of torsion angle molecular dynamics for efficient sampling of protein conformations.

We investigate the application of torsion angle molecular dynamics (TAMD) to augment conformational sampling of peptides and proteins. Interesting conformational changes in proteins mainly involve torsional degrees of freedom. Carrying out molecular dynamics in torsion space does not only explicitly sample the most relevant degrees of freedom, but also allows larger integration time steps with elimination of the bond and angle degrees of freedom. However, the covalent geometry needs to be fixed during internal coordinate dynamics, which can introduce severe distortions to the underlying potential surface in the extensively parameterized modern Cartesian-based protein force fields. A "projection" approach (Katritch et al. J Comput Chem 2003, 24, 254-265) is extended to construct an accurate internal coordinate force field (ICFF) from a source Cartesian force field. Torsion crossterm corrections constructed from local molecular fragments, together with softened van der Waals and electrostatic interactions, are used to recover the potential surface and incorporate implicit bond and angle flexibility. MD simulations of dipeptide models demonstrate that full flexibility in both the backbone phi/psi and side chain chi1 angles are virtually restored. The efficacy of TAMD in enhancing conformational sampling is then further examined by folding simulations of small peptides and refinement experiments of protein NMR structures. The results show that an increase of several fold in conformational sampling efficiency can be reliably achieved. The current study also reveals some complicated intrinsic properties of internal coordinate dynamics, beyond energy conservation, that can limit the maximum size of the integration time step and thus the achievable gain in sampling efficiency.

Computer Simulation↗

PubMed2005

Structure of the E. coli protein-conducting channel bound to a translating ribosome.

Secreted and membrane proteins are translocated across or into cell membranes through a protein-conducting channel (PCC). Here we present a cryo-electron microscopy reconstruction of the Escherichia coli PCC, SecYEG, complexed with the ribosome and a nascent chain containing a signal anchor. This reconstruction shows a messenger RNA, three transfer RNAs, the nascent chain, and detailed features of both a translocating PCC and a second, non-translocating PCC bound to mRNA hairpins. The translocating PCC forms connections with ribosomal RNA hairpins on two sides and ribosomal proteins at the back, leaving a frontal opening. Normal mode-based flexible fitting of the archaeal SecYEbeta structure into the PCC electron microscopy densities favours a front-to-front arrangement of two SecYEG complexes in the PCC, and supports channel formation by the opening of two linked SecY halves during polypeptide translocation. On the basis of our observation in the translocating PCC of two segregated pores with different degrees of access to bulk lipid, we propose a model for co-translational protein translocation.

Cell Membrane↗

PubMed2005

Detailed considerations for a balanced and broadly applicable force field: a study of substituted benzenes modeled with OPLS-AA.

Modern classical force fields have been traditionally parameterized by attempting to maximize agreement to any number of experimental and/or quantum mechanical target properties. As these force fields are pushed towards obtaining quantitative estimates of often subtle energetic differences, stringent and consistent parameterization criteria, particularly in regard to charge distributions, are required to ensure that systematic errors cancel, that parameters are transferable between molecules, and that performance does not significantly deteriorate when using more approximate methods, such as with continuum solvent models. Relative free energies of hydration are presented here for 40 mono- and disubstituted benzenes modeled with the OPLS-AA force field; heats of vaporization and pure liquid densities at standard conditions are presented when experimental data is available. Overall agreement between OPLS-AA and experiment is remarkable (average error = 0.5 kcal/mol for DeltaDeltaG(hydration), 1.0 kcal/mol for DeltaH(vap) (0), 0.02 g/mL for densities), yet several functional groups are identified as having consistent and correctable errors (alkyl-, nitro-, and thiobenzenes). Relative free energies of hydration obtained with rigorous free energy perturbations using explicit solvent are also compared with energies from minimizations using a generalized Born model (GB). There is high correlation between these estimates (R = 0.99), and as demonstrated here, reparameterization of the aforementioned groups can be guided with rapid GB calculations.

Algorithms↗