Forces and dynamics of glucose and inhibitor binding to sodium glucose co-transporter SGLT1 studied by single molecule force spectroscopy
Sprache des Titels:
gle molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic foce microscopy (AFM) tips carrying either thio-glucose, 2?-aminoethyl ?-D-glucopyranoside, or amino-phlorizin. Poly (ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thio-glucose (coupled via acrylamide to a long PEG), but decreased for amino-phlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucone binding sites for transport inhibitors have specific, temperature sensitive conformations.
Sprache der Kurzfassung:
Journal of Biological Chemistry
American Society for Biochemistry and Molecular Biology