High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex

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Combined UV and Liquid Scintillation Detection in HPLC. Formation and Depurination of Substitution Inert Pt(II) Complexes of Purine Nucleosides

Abstract

Combined UV- and liquid scintillation-HPLC has been applied to study the complexing of purine nucleosides with Pt(II)-diamine ions, and the effect of the complex formation on the acidic depurination.     

The Design and Characterization of Receptor-selective APRIL Variants

A proliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily with an important role in humoral immunity, is also implicated in several cancers as a prosurvival factor. APRIL binds two different TNF receptors, B cell maturation antigen (BCMA) and transmembrane activator and cylclophilin ligand interactor (TACI), and also interacts independently with heparan sulfate proteoglycans. Because APRIL shares binding of the TNF receptors with B cell activation factor, separating the precise signaling pathways activated by either ligand in a given context has proven quite difficult. In this study, we have used the protein design algorithm FoldX to successfully generate a BCMA-specific variant of APRIL, APRIL-R206E, and two TACI-selective variants, D132F and D132Y. These APRIL variants show selective activity toward their receptors in several in vitro assays. Moreover, we have used these ligands to show that BCMA and TACI have a distinct role in APRIL-induced B cell stimulation. We conclude that these ligands are useful tools for studying APRIL biology in the context of individual receptor activation.     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

BioMOCA—a Boltzmann transport Monte Carlo model for ion channel simulation

With the recent availability of high-resolution structural information for several key ion channel proteins and large-scale computational resources, Molecular Dynamics has become an increasingly popular tool for ion channel simulation. However, the CPU requirements for simulating ion transport on time scales relevant to conduction still exceed the resources presently available. To address this problem, we have developed Biology Monte Carlo (BioMOCA), a three-dimensional (3D) coarse-grained particle ion channel simulator based on the Boltzmann Transport Monte Carlo (BTMC) methodology. Although this approach is widely employed in the engineering community to study charge transport in electron devices, its application to molecular biology and electrolytes in general is new and hence must be validated. The pair correlation function, which is a measure of the microscopic structure of matter, provides a suitable benchmark to compare the BTMC method against the well-established Equilibrium Monte Carlo (EMC) approach. For validation purposes BioMOCA is used to simulate several simple homogeneous equilibrium electrolytes at concentrations of physiological interest. The ion–ion pair correlation functions computed from these simulations compare very well with those obtained from EMC simulations. We also demonstrate several performance-improving techniques that result in a several-fold speed-up without compromising the pair correlation function. BioMOCA is then used to perform full 3D simulations of ion transport in the gramicidin A channel in situ in a membrane environment, as well as to study the link between the electrostatic and dielectric properties of the protein and the channel's selectivity.