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. They found 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 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. The ligand binding biases the distribution of conformation by stabilizing the high-affinity receptor-competent structures. Computational predictions for the closely related ribose binding protein (RBP) show qualitatively similar results. 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. http://dx.doi.org/10.1529/biophysj.107.103911
The measurement of fluorescence from single protein molecules has become an important new tool in the study of dynamic processes, allowing for the direct visualization of the motions experienced by individual proteins and macromolecular complexes. The data from such single-molecule experiments are in the form of photon trajectories, consisting of arrival times and wavelength information on individual photons. The analysis of photon trajectories can be difficult, particularly if the motions are occurring at rates comparable to the photon arrival rate. In Andrec \emph{et al.} (2003) we introduced the use of HMMs for the analysis of photon trajectory data that operate using the photon data directly, without the need for ensemble averaging of the data as implied by correlation function analysis.