Jeffrey O. Boles

Brownian dynamics simulation of protein association

SummaryThe Brownian Dynamics (BD) method is applied to study the diffusive dynamics and interaction of two proteins, cytochrome c (CYTC) and cytochrome c peroxidase (CYP). We examine the role of protein electrostatic charge distribution in the facilitation of protein-protein docking prior to the electron transfer step, assessing the influence of individual charged amino acid residues. Accurate interaction potentials are computed by iterating the linearized Poisson-Boltzmann (PB) equation around the larger protein CYP. The low dielectric constant inside proteins, electrolyte screening effects and irregular protein surface topography are taken into account. We observe a large ensemble of electrostatically stable encounter complexes seemingly with acceptable geometric requirements for electron transfer rather than a single dominant complex. Stabilities of the large variety of docking complexes are rationalized in terms of generalized charged residue complementarities. However, it is found that the electrostatic interactions giving rise to complex stabilities are somewhat nonspecific in nature. A large series of additional simulations are performed in which individual charged residues on CYTC have been chemically modified. Resulting perturbations of the association rate are significant and qualitatively similar to results observed in comparable kinetics experiments. We therefore demonstrate the potential of the Brownian dynamics method to estimate the effects of site-directed mutagenesis on protein-protein and protein-ligand diffusional association rates.

The effect of dipole moment on diffusion controlled bimolecular reaction rates

Calculation of diffusion controlled bimolecular reaction rates for complicated macromolecular systems is made possible by a computer simulation approach based on the Brownian dynamics (BD) trajectory method. This is applied to the calculation of the diffusion controlled reaction rate between two charged spherical molecules, one of which represents a protein which possesses a strong dipole moment and asymmetric chemical reactivity. The role of the position of the dipole moment relative to reactive surfaces having various extents in media of varying ionic strength is assessed. No dipolar electrostatic effect on the diffusional rate constant of an isotropically reactive dipolar protein is observed. When the dipolar protein has a small axially symmetric reactive surface (e.g., of 10° extent), the dipole exerts a fivefold enhancement on the rate when the dipole vector is along the reactive patch axis, and retards the rate by a factor of 10 when oppositely disposed. A significant electrostatic effect persists t...

Isolation and characterization of pcp, a gene encoding a pyrrolidone carboxyl peptidase in Staphylococcus aureus

The pcp gene, encoding a pyrrolidone carboxyl peptidase (PYRase), was cloned from a lambda GT11 genomic library prepared from Staphylococcus aureus FDA 574 and sequenced. The pcp gene is located 740 bp downstream from cna, a gene that encodes a collagen-binding adhesin in S. aureus. S. aureus pcp encodes a 212-amino-acid (aa) polypeptide. The pcp gene was overexpressed in Escherichia coli and the PYRase purified to homogeneity. The recombinant enzyme exhibited biological activity, as determined using the chromogenic substrate L-pyroglutamyl-beta-napthylamide. Biochemical analysis of the PYRase using thiol-blocking chemicals suggested that the enzyme belongs to the cysteine peptidase family. Moreover, multiple sequence alignment revealed a high degree of similarity to previously described bacterial PYRases. This family of peptidases has been used to selectively remove the N-terminal pyrrolidone carboxylic acid residue found on certain blocked proteins and peptides prior to aa sequencing. However, the exact biological role of PYRases has yet to be elucidated.

Synthesis and incorporation of [6,7]-selenatryptophan into dihydrofolate reductase.

Until recently, the only selenium containing amino acid which could be used to completely substitute for a wild type amino acid was selenomethionine (SeMet). In the last decade the preparation of SeMet containing proteins has proved to be valuable tools in the determination of three-dimensional structure by multiwavelength anomalous diffraction (MAD) techniques. The potential utility of a selenium containing tryptophan analog, beta-seleno[3,2-b]pyrrolyl-L-alanine ([4,5]selenatryptophan), has recently been demonstrated in the literature. This finding shows promise for the bioincorporation of its positional isomer, beta-selenolo[2,3-b]pyrrolyl-L-alanine ([6,7]selenatryptophan), thereby adding to the essential arsenal of selenium-containing amino acids for use in the characterization of proteins. The synthesis of [6,7]selenatryptophan by enzymatic biotransformation with tryptophan synthase from selenolo[2,3-b]pyrrole was carried out as well as its characterization by NMR spectroscopy and thin layer chromatography. Selenatryptophyl dihydrofolate reductase ([6,7]SeTrp-DHFR) was then synthesized in vivo, purified, and found to exhibit no perturbations to enzymatic activity.

Wipe sampling of amphetamine-type stimulants and recreational drugs on selected household surfaces with analysis by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Sorption characteristics of eight drugs related to recreational and clandestine activity-amphetamine, cocaine, heroin, N-formyl amphetamine, N-formyl methamphetamine, methamphetamine, 3, 4-methylenedioxymethamphetamine (MDMA), and pseudoephedrine-were evaluated on selected kitchen countertop surfaces. Methanol-dampened Whatman 40 filter paper wipes were used to collect samples from eleven surfaces including alkyd resin, ceramic tiles, glass, granite, laminate, limestone, marble, quartz compac, quartz real, soap stone, and stainless steel. The filter paper wipes were analyzed by a rapid three-minute UPLC-QTOF method, following ammonium acetate buffer (pH 5.8-6.2) extraction. The average percentage recoveries after 15 h of exposure to the surface materials tested, was found to be highest for cocaine and MDMA and lowest for amphetamine and methamphetamine. Among the eleven countertop surfaces, overall recoveries for marble were observed to be the least, whereas soapstone, quartz compac and stainless steel were among the highest. Scanning electron microscopic images of the surfaces provided a unique view of surface irregularities that potentially influenced drug recovery. Aging, migration, solvent composition, and volatility were examined. The variation in recovery of drugs was attributed to four key factors: compound volatility, surface composition, surface-compound interaction, and solvent composition.