Photoaffinity labeling techniques have been used for decades to identify drug binding sites and to study the structural biology of allosteric transitions in transmembrane proteins including pentameric ligand-gated ion channels (pLGIC). In a typical photoaffinity labeling experiment, to identify drug binding sites, UV light is used to introduce a covalent bond between a photoreactive ligand (which upon irradiation at the appropriate wavelength converts to a reactive intermediate) and amino acid residues that lie within its binding site. Then protein chemistry and peptide microsequencing techniques are used to identify these amino acids within the protein primary sequence. These amino acid residues are located within homology models of the receptor to identify the binding site of the photoreactive probe. Molecular modeling techniques are then used to model the binding of the photoreactive probe within the binding site using docking protocols. Photoaffinity labeling directly identifies amino acids that contribute to drug binding sites regardless of their location within the protein structure and distinguishes them from amino acids that are only involved in the transduction of the conformational changes mediated by the drug, but may not be part of its binding site (such as those identified by mutational studies). Major limitations of photoaffinity labeling include the availability of photoreactive ligands that faithfully mimic the properties of the parent molecule and protein preparations that supply large enough quantities suitable for photoaffinity labeling experiments. When the ligand of interest is not intrinsically photoreactive, chemical modifications to add a photoreactive group to the parent drug, and pharmacological evaluation of these chemical modifications become necessary. With few exceptions, expression and affinity-purification of proteins are required prior to photolabeling. Methods to isolate milligram quantities of highly enriched pLGIC suitable for photoaffinity labeling experiments have been developed. In this chapter, we discuss practical aspects of experimental strategies to identify allosteric modulator binding sites in pLGIC using photoaffinity labeling.

Introduction: Pharmacy fellowships are post-doctoral training programs intended to prepare pharmacy graduates for careers in research or the pharmaceutical industry. There are currently 131 pharmacy fellowship programs in the United States, but standardization, interest among students, and overall research regarding these fellowships are ambiguous at best. This literature review was conducted to describe common facilitators, challenges, contents and outcomes of fellowships, and to evaluate the group of programs as a whole.

It is well-understood that cell membranes are crowded and complex environments, containing up to 50 % protein by mass [1] and comprised of myriad types of lipid species [2]. Less well understood is the molecular detail of the effects of complexity and crowding on membrane organisation and dynamics. Advances in coarse-grained force fields and computational power mean that large-scale coarse-grained (CG) molecular dynamics (MD) simulations are increasingly being used to gain such understanding [3].