The role of protein dynamics on different time scales in enzyme catalysis remains an area of active debate. The connection between enzyme dynamics on the femtosecond time scale and transition state formation has been demonstrated in human purine nucleoside phosphorylase (PNP) through the study of a mass-altered enzyme. Isotopic substitution in human PNP (heavy PNP) decreased the rate of on-enzyme chemistry but did not alter either the transition state structure or steady-state kinetic parameters. Here we investigate the underlying atomic motions associated with altered barrier crossing probability for heavy PNP. Transition path sampling was employed to illuminate the molecular differences between barrier crossing in light and heavy enzymes. The mass effect is apparent in promoting vibrations that polarize the N-ribosidic bond, and that promote the stability of the purine leaving group. These motions facilitate barrier crossing.

Genome-Wide Association Studies (GWAS), whole genome sequencing, and high-throughput omics techniques have generated vast amounts of genotypic and molecular phenotypic data. However, these data have not yet been fully explored to improve the effectiveness and efficiency of drug discovery, which continues along a one-drug-one-target-one-disease paradigm. As a partial consequence, both the cost to launch a new drug and the attrition rate are increasing. Systems pharmacology and pharmacogenomics are emerging to exploit the available data and potentially reverse this trend, but, as we argue here, more is needed. To understand the impact of genetic, epigenetic, and environmental factors on drug action, we must study the structural energetics and dynamics of molecular interactions in the context of the whole human genome and interactome. Such an approach requires an integrative modeling framework for drug action that leverages advances in data-driven statistical modeling and mechanism-based multiscale modeling and transforms heterogeneous data from GWAS, high-throughput sequencing, structural genomics, functional genomics, and chemical genomics into unified knowledge. This is not a small task, but, as reviewed here, progress is being made towards the final goal of personalized medicines for the treatment of complex diseases.

The interactions of the 50S subunit of bacterial ribosome with antibiotic sparsomycin (SPS) and five analogs (AN) are investigated through the calculation of the standard (absolute) binding free energy and the characterization of conformational dynamics. The standard binding free energies of the complexes are computed using free energy perturbation molecular dynamics (FEP/MD) simulations with explicit solvent. Restraining potentials are applied and then released during the simulation to efficiently sample the changes in translational, orientational, and conformational freedom of the ligand and receptor upon binding. The biasing effects of the restraining potentials are rigorously removed. The loss of conformational freedom of the ligand upon binding is determined by introducing a potential of mean force (PMF) as a function of the root-mean-square deviation (rmsd) of the ligand relative to its conformation in the bound state. To reduce the size of the simulated system, the binding pocket of the ribosome is simulated in the framework of the generalized solvent boundary potential (GSBP). The number of solvent molecules in the buried binding site is treated via grand canonical Monte Carlo (GCMC) during the FEP/MD simulations. The correlation coefficient between the calculated and measured binding free energies is 0.96, and the experimentally observed ranking order for the binding affinities of the six ligands is reproduced. However, while the calculated affinities of the strong binders agree well with the experimental values, those for the weak binders are underestimated.

Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. ␤-Lactam antibiotics normally interfere with this process by reacting covalently with the active site serine to form a stable acyl-enzyme. The design of novel ␤-lactams active against penicillin-susceptible and penicillin-resistant organisms will require a better understanding of the molecular details of this reaction. To that end, we compared the affinities of different ␤-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (⌬1-36 rPBP5). The soluble protein, ⌬1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH 2 -terminal protein sequence was verified by amino acid sequencing. Using ␤-lactams with different R1 side chains, we show that azlocillin has greater affinity for ⌬1-36 rPBP5 than piperacillin and ampicillin (apparent K i ‫؍‬ 7 ؎ 0.3 M, compared to 36 ؎ 3 and 51 ؎ 10 M, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k 2 ‫؍‬ 15 ؎ 4 M ؊1 s ؊1 ). Meropenem demonstrates an affinity for ⌬1-36 rPBP5 comparable to that of ampicillin (apparent K i ‫؍‬ 51 ؎ 15 M) but is slower at acylating (apparent k 2 ‫؍‬ 0.14 ؎ 0.02 M ؊1 s ؊1 ). This characterization defines important structure-activity relationships for this clinically relevant type II transpeptidase, shows that the rate of formation of the acyl-enzyme is an essential factor determining the efficacy of a ␤-lactam, and suggests that the specific side chain interactions of ␤-lactams could be modified to improve inactivation of resistant PBPs.Bacterial penicillin-binding proteins (PBPs) are members of the penicilloyl serine transferase family of enzymes (2,3,8,11). PBPs catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from the glycolipid precursor, lipid II (2). These reactions are composed of two parts: transglycosylation and transpeptidation. Transglycosylation joins the disaccharides of lipid II monomers to the peptidoglycan, creating the N-acetylmuramic acid and N-acetylglucosamine backbone of the murein sacculus. Transpeptidation cross-links the glycosidic backbone into a network of interlocking units that confers exceptional tensile strength. There are three broad types of PBPs (types I to III). Type I PBPs are multifunctional, multidomain proteins catalyzing both peptidoglycan polymerization and cross-linking. Unfortunately, penicillin resistance mediated by PBPs that are, or have become, insensitive to the inhibition by ␤-lactams is found in many clinically important pathogens. Some of the more problematic pathogens are Staphylococcus aureus (methicillinresistant S. aureus [MRSA]), coagulase-negative staphylococci (CNS), Neisseria gonorrhoeae, Streptococcus pneumoniae, Enterococcus faecium, and Enterococcus faecalis (6). The increasing prevalence and resistance of these pathogens are overcoming our best therapeutic efforts, since few antibiotics are available to treat these penicillin-resistant o...

The standard (absolute) binding free energy of the antibiotic sparsomycin with the 50S bacteria ribosomal subunit is calculated using molecular dynamics (MD) free energy perturbation (FEP) simulations with restraining potentials developed by Wang et al. [Biophys. J. 91:2798-2814 (2006)]. In the simulation protocol, restraining potentials are activated for the orientational and translational movements of the ligand relative to the binding site when it is decoupled from the binding pocket, and then released once the ligand fully interacts with the rest of the system. A reduced system is simulated to decrease the computational cost of the FEP/MD calculations and the effects of the surrounding atoms are incorporated using the generalized solvent boundary potential (GSBP) method. The loss of conformational freedom of the ligand upon binding is characterized using the potential of mean force (PMF) as a function of the root-mean-square deviation (RMSD) relative to the bound conformation. The number of water molecules in the binding pocket is allowed to fluctuate dynamically in response to the ligand during the calculations by combining FEP/MD with grand canonical Monte Carlo (GCMC) simulations. The calculated binding free energy is about -6 kcal/mol, which is in reasonable agreement with the experimental value. The information gleaned from this study provides new insight on the recognition of ribosome by sparsomycin and highlights the challenges in calculations of absolute binding free energies in these systems.

Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long-range proton translocation between the central (the retinal Schiff base SB216, D85, and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation states of the SB216, D85, D212, and E204. The side-chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side-chain reorientation of R82 modulates the hydrogen-bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton-transfer in the methyl guanidinium-hydronium-hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the bacteriorhodopsin models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O' state where the proton on D85 is transferred to D212.

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A new spectrofluorimetric method was developed for the determination of trace amounts of coenzyme A (CoA). In the presence of periodic acid (H5IO6), CoA can remarkably enhance the fluorescence intensity of the Tb3+-ciprofloxacin (CIP) complex at 545 nm in a buffer solution at pH 5.4; the enhanced fluorescence intensity of the Tb3+ ion is proportional to the concentration of CoA. The optimal conditions for the determination of CoA were also investigated. The linear range and the detection limit for the determination of CoA were 6.08x10(-6)-1.64x10(-5) and 2.1x10(-8) mol L-1, respectively. This method is simple, practical and relatively free of interference from coexisting substances, and can be successfully applied to assess CoA in injection and biological samples. Moreover, the enhancement mechanism of the fluorescence intensity of the CoA-Tb3+-CIP system in the presence of H5IO6 is also discussed.