Glucose/Galactose Binding Protein

Enteric bacteria use glucose/galactose binding protein (GGBP) in separate pathways to actively transport methylgalactosides across the cell membrane and to chemically sense them as part of the swimming regulatory scheme. Crystallographic and bulk steady-state experiments have been reported for GGBP. Binding of glucose has been described both by a single and by multiple binding constants. GGBP undergoes large dynamic structural fluctuations that are decreased, but not eliminated upon binding of glucose. Thermodynamic characterisation of the structural changes associated with ligand recognition and protein-complex docking can be difficult and detailed understanding of the role of conformation in ligand binding and delivery to the cytosol or activation of the methyl accepting chemotaxis protein Trg are not well characterised.

Here we show that GGBP fluctuates between at least three conformations with the relative weights of those conformations being modulated by the binding of glucose. Each structure has different binding affinity and thermodynamic properties. The single binding site of GGBP was considered to have a single association constant, our results suggest that the binding constant is conformationally dependent. Moreover, the ligand binding does not induce the conformational change, rather, it biases the distribution of conformation by stabilising the high-affinity receptor-competent structures. Computational predictions for the closely related ribose binding protein show qualitatively similar results.12 The presence of two high-affinity binding structures is suggestive of the different membrane receptors to which GGBP must bind to provide either active transport or chemotaxis. Conformational plasticity is increasingly becoming recognised as an important issue in drug resistance. GGBP is an ABC transporter system which are common targets for therapeutics. An inhibitor for flexible targets like GGBP would need to interact with all parts of the binding-competent conformational ensemble. Glucose/galactose binding protein (GGBP) is a component of the the β-methyl galactoside chemosensory transport system in Escherichia coli responsible for delivery of glucose and galactose to periplasmic membrane receptor proteins. Previous studies of GGBP have shown large-scale conformational changes upon ligand binding.

We discuss results of bulk steady-state and time-resolved fluorescence measurements of this protein. The protein has a point mutation at residue Leucine 255 to Cysteine (L255C) and is conjugated with acrylodan. The N-terminus is labeled with a long-lived (~ 1 µs) ruthenium complex. Using a two-state binding model to analyze fluorescence anisotropy data and fitting the data globally over the ligand titration, we are able to extract time scales for local dye reorientation, rotational diffusion of the protein, and ligand binding constant. The results indicate up to 40% concentration of the "closed" (glucose-bound) protein conformation exist at 0 M glucose, evidence that proteins are continuously sampling their thermodynamically available conformational space. Global analysis of the fluorescence lifetime, time-resolved anisotropy, and dynamic Stokes shift indicates that there are three distinct states of the protein the free energy of which is modified by the binding of glucose. This suggests a dynamic conformational sampling model of ligand binding where the ligand stabilizes the closed conformation(s) with respect to the other conformation(s). This picture requires barriers of in the apo form of the protein with the barrier to opening increasing due to the stabilization of the closed state(s). This result confirms a theoretical prediction made on a related system, ribose binding protein.

In collaboration with the group of Ron Levy we have begun umbrella-potential sampling and replica exchange molecular dynamics simulations of GGBP to better characterize the heterogeneity of structures associated with the holo and apo forms of the protein.