Photoaffinity labeling is used to covalently attach ligands to macromolecules to determine their spatial arrangement and structure. Benzophenone (BP) groups are widely used for covalent photoaffinity labeling and for probing protein interactions. We developed bifunctional BP photoactivatable derivatives using three different general chemical approaches. In addition to the photoaffinity reactivity of the BP, these derivatives contain an additional group: A radioactive tracer for biological studies, or an N-ethylmaleimide group as an additional crosslinker, or a biotin group to be used during purification and characterization of probe-protein complexes using the high-affinity biotin-avidin interaction. A model series of photoaffinity labeling probes was synthesized based on the arbutin ligand. These compounds can be used as probes to study the arbutin binding site of microbial beta-glucoside transporters by photolabeling residues in its vicinity. The second functionality provides additional options for studying proteins and binding sites. The probes were developed using different methodologies: (i) a diazotation reaction; (ii) protecting group methodology; and (iii) solid-phase synthesis. These procedures are general and provide a simple and versatile approach for synthesizing bifunctional BP ligands, as demonstrated here on arbutin.
The Escherichia coli BglF protein, a sugar permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), catalyzes concomitant transport and phosphorylation of -glucosides across the cytoplasmic membrane. Despite intensive studies of PTS permeases, the mechanism that couples sugar translocation to phosphorylation and the nature of the translocation apparatus are poorly understood. Like many PTS permeases, BglF consists of a transmembrane domain, which in addition to transmembrane helices (TMs) contains a big cytoplasmic loop and two hydrophilic domains, one containing a conserved cysteine that phosphorylates the incoming sugar. We previously reported that the big hydrophilic loop, which connects TM VI to TM VII, contains regions that alternate between facing-in and facing-out states and speculated that it is involved in creating the sugar translocation channel. In the current study we used [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), a membrane-impermeative thiol-specific reagent, to identify sites that are involved in sugar transport. These sites map to the regions that border the big loop. Using cross-linking reagents that penetrate the cell, we could demonstrate spatial proximity between positions at the center of the big loop and the phosphorylation site, suggesting that the two regions come together to execute sugar phosphotransfer. Additionally, positions on opposite ends of the big loop were found to be spatially close. Cys accessibility analyses suggested that the sugar induces a change in this region. Taken together, our results demonstrate that the big loop participates in creating the sugar pathway and explain the observed coupling between translocation of PTS sugars from the periplasm to the cytoplasm and their phosphorylation.The phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) is a central system in bacteria that controls preferential use of carbon sources. In addition to catabolite repression and inducer exclusion, the PTS is responsible for the translocation of a variety of energetically favorable carbohydrates across the cytoplasmic membrane and for their concomitant phosphorylation (10). It consists of two general PTS proteins, enzyme I and HPr, and a number of sugar-specific permeases. The phosphorylation cascade starts with enzyme I, which accepts a phosphoryl group from phosphoenolpyruvate and passes it on to HPr; the latter molecule phosphorylates the sugar-specific permeases. BglF (also designated EII bgl ) is a PTS permease that catalyzes phosphotransfer of -glucosides into Escherichia coli cells (13). Like many other PTS permeases, BglF is composed of three conserved domains (for reviews, see references 27 and 22): the hydrophilic A and B domains and the membrane C domain (corresponding to the IIA bgl , IIB bgl , and IIC bgl domains, respectively). The A domain is phosphorylated at H547 by HPr; the phosphate is then transferred to C24 in the B domain and subsequently to the incoming -glucosides (8, 28). In addition, BglF ...
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