Biomedical subjects

J A McCammon

Publications and source records attributed to J A McCammon.

At least 19 recordsLinked to original sources

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

A minimal model for stabilization of biomolecules by hydrocarbon cross-linking.

Programmed cell death regulating protein motifs play an essential role in the development of an organism, its immune response, and disease-related cellular mechanisms. Among those motifs the BH3 domain of the BCL-2 family is found to be of crucial importance. Recent experiments showed how the isolated, otherwise unstructured BH3 peptide can be modified by a hydrocarbon linkage to regain function. We parametrized a reduced, dynamic model for the stability effects of such covalent cross-linking and confirmed that the model reproduces the reinforcement of the structural stability of the BH3 motif by cross-linking. We show that an analytically solvable model for thermostability around the native state is not capable of reproducing the stabilization effect. This points to the crucial importance of the peptide dynamics and the fluctuations neglected in the analytic model for the cross-linking system to function properly. This conclusion is supported by a thorough analysis of a simulated Go model. The resulting model is suitable for rational design of generic cross-linking systems in silicio.

Algorithms↗

PubMed2006

E230Q mutation of the catalytic subunit of cAMP-dependent protein kinase affects local structure and the binding of peptide inhibitor.

The active site of the mammalian cAMP-dependent protein kinase catalytic subunit (C-subunit) has a cluster of nonconserved acidic residues-Glu127, Glu170, Glu203, Glu230, and Asp241-that are crucial for substrate recognition and binding. Studies have shown that the Glu230 to Gln mutant (E230Q) of the enzyme has physical properties similar to the wild-type enzyme and has decreased affinity for a short peptide substrate, Kemptide. However, recent experiments intended to crystallize ternary complex of the E230Q mutant with MgATP and protein kinase inhibitor (PKI) could only obtain crystals of the apo-enzyme of E230Q mutant. To deduce the possible mechanism that prevented ternary complex formation, we used the relaxed-complex method (Lin, J.-H., et al. J Am Chem Soc 2002, 24, 5632-5633) to study PKI binding to the E230Q mutant C-subunit. In the E230Q mutant, we observed local structural changes of the peptide binding site that correlated closely to the reduced PKI affinity. The structural changes occurred in the F-to-G helix loop and appeared to hinder PKI binding. Reduced electrostatic potential repulsion among Asp241 from the helix loop section and the other acidic residues in the peptide binding site appear to be responsible for the structural change.

Binding Sites↗

PubMed2006

Coupling hydrophobicity, dispersion, and electrostatics in continuum solvent models.

An implicit solvent model is presented that couples hydrophobic, dispersion, and electrostatic solvation energies by minimizing the system Gibbs free energy with respect to the solvent volume exclusion function. The solvent accessible surface is the output of the theory. The method is illustrated with the solvation of simple solutes on different length scales and captures the sensitivity of hydration to the particular form of the solute-solvent interactions in agreement with recent computer simulations.

Alkanes↗

PubMed2006

Coupling nonpolar and polar solvation free energies in implicit solvent models.

Recent studies on the solvation of atomistic and nanoscale solutes indicate that a strong coupling exists between the hydrophobic, dispersion, and electrostatic contributions to the solvation free energy, a facet not considered in current implicit solvent models. We suggest a theoretical formalism which accounts for coupling by minimizing the Gibbs free energy of the solvent with respect to a solvent volume exclusion function. The resulting differential equation is similar to the Laplace-Young equation for the geometrical description of capillary interfaces but is extended to microscopic scales by explicitly considering curvature corrections as well as dispersion and electrostatic contributions. Unlike existing implicit solvent approaches, the solvent accessible surface is an output of our model. The presented formalism is illustrated on spherically or cylindrically symmetrical systems of neutral or charged solutes on different length scales. The results are in agreement with computer simulations and, most importantly, demonstrate that our method captures the strong sensitivity of solvent expulsion and dewetting to the particular form of the solvent-solute interactions.

Computer Simulation↗

PubMed2005

Substrate concentration dependence of the diffusion-controlled steady-state rate constant.

The Smoluchowski approach to diffusion-controlled reactions is generalized to interacting substrate particles by including the osmotic pressure and hydrodynamic interactions of the nonideal particles in the Smoluchoswki equation within a local-density approximation. By solving the strictly linearized equation for the time-independent case with absorbing boundary conditions, we present an analytic expression for the diffusion-limited steady-state rate constant for small substrate concentrations in terms of an effective second virial coefficient B2*. Comparisons to Brownian dynamics simulations excluding hydrodynamic interactions show excellent agreement up to bulk number densities of B2*rho0 < approximately = 0.4 for hard sphere and repulsive Yukawa-like interactions between the substrates. Our study provides an alternative way to determine the second virial coefficient of interacting macromolecules experimentally by measuring their steady-state rate constant in diffusion-controlled reactions at low densities.

Anisotropy↗

PubMed2001

Kinetic mechanism of end-to-end annealing of actin filaments.

We investigated the effect of actin filament length and capping protein on the rate of end-to-end annealing of actin filaments. Long filaments were fragmented by shearing and allowed to recover. Stabilizing filaments with phalloidin in most experiments eliminated any contribution of subunit dissociation and association to the redistribution of lengths but did not affect the results. Two different assays, fluorescence microscopy to measure filament lengths and polymerization to measure concentration of barbed filament ends, gave the same time-course of annealing. The rate of annealing declines with time as the average filament length increases. Longer filaments also anneal slower than short filaments. The second-order annealing rate constant is inversely proportional to mean polymer length with a value of 1.1 mM(-1) s(-1)/length in subunits. Capping protein slows but does not prevent annealing. Annealing is a highly favorable reaction with a strong influence on the length of polymers produced by spontaneous polymerization and should be considered in thinking about polymer dynamics in cells.

Actin Cytoskeleton↗

PubMed2001

Atomistic Brownian dynamics simulation of peptide phosphorylation.

We report the implementation of an all-atom Brownian dynamics simulation model of peptides using the constraint algorithm LINCS. The algorithm has been added as a part of UHBD. It uses adaptive time steps to achieve a balance between computational speed and stability. The algorithm was applied to study the effect of phosphorylation on the conformational preference of the peptide Gly-Ser-Ser-Ser. We find that the middle serine residue experiences considerable conformational change from the C(7eq) to the alpha(R) structure upon phosphorylation. NMR (3)J coupling constants were also computed from the Brownian trajectories using the Karplus equation. The calculated (3)J results agree reasonably well with experimental data for phosphorylated peptide but less so for doubly charged phosphorylated one.

Algorithms↗

PubMed2001

Ordered water and ligand mobility in the HIV-1 integrase-5CITEP complex: a molecular dynamics study.

A 2 ns molecular dynamics simulation has been carried out for the HIV-1 integrase-5CITEP complex in order to understand the role of water in defining the ligand's binding mode and to address issues of binding site flexibility and ligand motion. Although the ligand retains considerable mobility within the active site, a structural water molecule bridging 5CITEP with Asp 64 and Asn 155 is identified in the simulation. Consideration of this water molecule could open a route to new HIV-1 integrase inhibitors.

Binding Sites↗

PubMed2001

Electrostatics of nanosystems: application to microtubules and the ribosome.

Evaluation of the electrostatic properties of biomolecules has become a standard practice in molecular biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calculations to relatively small biomolecular systems. Here we present the application of numerical methods to enable the trivially parallel solution of the Poisson-Boltzmann equation for supramolecular structures that are orders of magnitude larger in size. As a demonstration of this methodology, electrostatic potentials have been calculated for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.

Algorithms↗

PubMed2001

Computational analysis of PKA-balanol interactions.

Protein kinases are important targets for designing therapeutic drugs. This paper illustrates a computational approach to extend the usefulness of a single protein-inhibitor structure in aiding the design of protein kinase inhibitors. Using the complex structure of the catalytic subunit of PKA (cPKA) and balanol as a guide, we have analyzed and compared the distribution of amino acid types near the protein-ligand interface for nearly 400 kinases. This analysis has identified a number of sites that are more variable in amino acid types among the kinases analyzed, and these are useful sites to consider in designing specific protein kinase inhibitors. On the other hand, we have found kinases whose protein-ligand interfaces are similar to that of the cPKA-balanol complex and balanol can be a useful lead compound for developing effective inhibitors for these kinases. Generally, this approach can help us discover new drug targets for an existing class of compounds that have already been well characterized pharmacologically. The relative significance of the charge/polarity of residues at the protein-ligand interface has been quantified by carrying out computational sensitivity analysis in which the charge/polarity of an atom or functional group was turned off/on, and the resulting effects on binding affinity have been examined. The binding affinity was estimated by using an implicit-solvent model in which the electrostatic contributions were obtained by solving the Poisson equation and the hydrophobic effects were accounted for by using surface-area dependent terms. The same sensitivity analysis approach was applied to the ligand balanol to develop a pharmacophoric model for searching new drug leads from small-molecule libraries. To help evaluate the binding affinity of designed inhibitors before they are made, we have developed a semiempirical approach to improve the predictive reliability of the implicit-solvent binding model.

Azepines↗

PubMed2001

A model for enzyme-substrate interaction in alanine racemase.

We report on a theoretical model for the complex of the enzyme alanine racemase with its natural substrate (L-alanine) and cofactor (pyridoxal 5'-phosphate). Electrostatic potentials were calculated and ionization states were predicted for all of the ionizable groups in alanine racemase. Some rather unusual charge states were predicted for certain residues. Tyr265' has an unusually low predicted pK(a) of 7.9 and at pH 7.0 has a predicted average charge of -0.37, meaning that 37% of the Tyr265' residues in an ensemble of enzyme molecules are in the phenolate form. At pH 8-9, the majority of Tyr265' side groups will be in the phenolate form. This lends support to the experimental evidence that Tyr265' is the catalytic base involved in the conversion of L-alanine to D-alanine. Residues Lys39 and Lys129 have predicted average charges of +0.91 and +0.14, respectively, at pH 7.0. Lys39 is believed to be the catalytic base for the conversion of D-alanine to L-alanine, and the present results show that, at least some of the time, it is in the unprotonated amine form and thus able to act as a base. Cys311', which is located very close to the active site, has an unusually low predicted pK(a) of 5.8 and at pH 7.0 has a predicted average charge of -0.72. The very low predicted charge for Lys129 is consistent with experimental evidence that it is carbamylated, since an unprotonated amine group is available to act as a Lewis base and form the carbamate with CO(2). Repeating the pK(a) calculations on the enzyme with Lys129 in carbamylated form predicts trends similar to those of the uncarbamylated enzyme. It appears that the enzyme has the ability to stabilize negative charge in the region of the active site. Implications for selective inhibitor design are discussed.

Alanine Racemase↗

PubMed2001

Statistical analysis of the fractal gating motions of the enzyme acetylcholinesterase.

The enzyme acetylcholinesterase has an active site that is accessible only by a "gorge" or main channel from the surface, and perhaps by secondary channels such as the "back door." Molecular-dynamics simulations show that these channels are too narrow most of the time to admit substrate or other small molecules. Binding of substrates is therefore "gated" by structural fluctuations of the enzyme. Here, we analyze the fluctuations of these possible channels, as observed in the 10.8-ns trajectory of the simulation. The probability density function of the gorge proper radius (defined in the text) was calculated. A double-peak feature of the function was discovered and therefore two states with a threshold were identified. The relaxation (transition probability) functions of these two states were also calculated. The results revealed a power-law decay trend and an oscillation around it, which show properties of fractal dynamics with a "complex exponent." The cross correlation of potential energy versus proper radius was also investigated. We discuss possible physical models behind the fractal protein dynamics; the dynamic hierarchical model for glassy systems is evaluated in detail.

Acetylcholinesterase↗

PubMed2001

Identification of protein oligomerization states by analysis of interface conservation.

The discrimination of true oligomeric protein-protein contacts from nonspecific crystal contacts remains problematic. Criteria that have been used previously base the assignment of oligomeric state on consideration of the area of the interface and/or the results of scoring functions based on statistical potentials. Both techniques have a high success rate but fail in more than 10% of cases. More importantly, the oligomeric states of several proteins are incorrectly assigned by both methods. Here we test the hypothesis that true oligomeric contacts should be identifiable on the basis of an increased degree of conservation of the residues involved in the interface. By quantifying the degree of conservation of the interface and comparing it with that of the remainder of the protein surface, we develop a new criterion that provides a highly effective complement to existing methods.

Models, Molecular↗

PubMed2001

Thermodynamics and kinetics of actin filament nucleation.

We have performed computer simulations and free energy calculations to determine the thermodynamics and kinetics of actin nucleation and thus identify a probable nucleation pathway and critical nucleus size. The binding free energies of structures along the nucleation pathway are found through a combination of electrostatic calculations and estimates of the entropic and surface area contributions. The association kinetics for the formation of each structure are determined through a series of Brownian dynamics simulations. The combination of the binding free energies and the association rate constants determines the dissociation rate constants, allowing for a complete characterization of the nucleation and polymerization kinetics. The results indicate that the trimer is the size of the critical nucleus, and the rate constants produce polymerization plots that agree very well with experimental results over a range of actin monomer concentrations.

Actin Cytoskeleton↗

PubMed2001

Analysis of a 10-ns molecular dynamics simulation of mouse acetylcholinesterase.

A 10-ns molecular dynamics simulation of mouse acetylcholinesterase was analyzed, with special attention paid to the fluctuation in the width of the gorge and opening events of the back door. The trajectory was first verified to ensure its stability. We defined the gorge proper radius as the measure for the extent of gorge opening. We developed an expression of an inter-atom distance representative of the gorge proper radius in terms of projections on the principal components. This revealed the fact that collective motions of many scales contribute to the opening behavior of the gorge. Covariance and correlation results identified the motions of the protein backbone as the gorge opens. In the back-door region, side-chain dihedral angles that define the opening were identified.

Acetylcholinesterase↗

PubMed2001

Calculation of weak protein-protein interactions: the pH dependence of the second virial coefficient.

Interactions between proteins are often sufficiently weak that their study through the use of conventional structural techniques becomes problematic. Of the few techniques capable of providing experimental measures of weak protein-protein interactions, perhaps the most useful is the second virial coefficient, B(22), which quantifies a protein solution's deviations from ideal behavior. It has long been known that B(22) can in principle be computed, but only very recently has it been demonstrated that such calculations can be performed using protein models of true atomic detail (Biophys. J. 1998, 75:2469-2477). The work reported here extends these previous efforts in an attempt to develop a transferable energetic model capable of reproducing the experimental trends obtained for two different proteins over a range of pH and ionic strengths. We describe protein-protein interaction energies by a combination of three separate terms: (i) an electrostatic interaction term based on the use of effective charges, (ii) a term describing the electrostatic desolvation that occurs when charged groups are buried by an approaching protein partner, and (iii) a solvent-accessible surface area term that is used to describe contributions from van der Waals and hydrophobic interactions. The magnitude of the third term is governed by an adjustable, empirical parameter, gamma, that is altered to optimize agreement between calculated and experimental values of B(22). The model is applied separately to the proteins lysozyme and chymotrypsinogen, yielding optimal values of gamma that are almost identical. There are, however, clear difficulties in reproducing B(22) values at the extremes of pH. Explicit calculation of the protonation states of ionizable amino acids in the 200 most energetically favorable protein-protein structures suggest that these difficulties are due to a neglect of the protonation state changes that can accompany complexation. Proper reproduction of the pH dependence of B(22) will, therefore, almost certainly require that account be taken of these protonation state changes. Despite this problem, the fact that almost identical gamma values are obtained from two different proteins suggests that the basic energetic formulation used here, which can be evaluated very rapidly, might find use in dynamical simulations of weak protein-protein interactions at intermediate pH values.

Biophysical Phenomena↗

PubMed2001

The structure of Sky1p reveals a novel mechanism for constitutive activity.

Sky1p is the only member of the SR protein kinase (SRPK) family in Saccharomyces cerevisiae. SRPKs are constitutively active kinases that display remarkable substrate specificity and have been implicated in RNA processing. Here we present the three-dimensional structure of a fully active truncated Sky1p. Analysis of the structure and structure-based functional studies reveal that the C-terminal tail, an unusual Glu residue located in the P+1 loop, and a unique mechanism for the positioning of helix alpha C act together to render Sky1p constitutively active. We have modeled a substrate peptide bound to Sky1p. The modeled complex combined with mutagenesis studies illustrate the molecular basis for substrate recognition by this kinase and suggest a mechanism by which SRPKs catalyze a sequential phosphorylation reaction of the consecutive RS dipeptide repeats characteristic of mammalian SRPK substrates.

Amino Acid Sequence↗