title: Forces and dynamics of glucose and inhibitor binding to sodium glucose co-transporter SGLT1 studied by single molecule force spectroscopy.
abstract: Single 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 force microscopy tips carrying either thioglucose, 2'-aminoethyl β-d-glucopyranoside, or aminophlorizin. 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 thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin 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 aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.
authors: From the Institute for Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria.
authors: Isabel
authors: I
authors: Neundlinger
authors: From the Institute for Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, Department of Biology, Faculty of Science, Mahidol University and Nanotec-MU Center of Excellence on Intelligent Materials and Systems, 272 Rama VI, Ratchathewi, Bangkok 10400, Thailand.
authors: Theeraporn
authors: T
authors: Puntheeranurak
authors: From the Institute for Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria.
authors: Linda
authors: L
authors: Wildling
authors: Agilent Technologies, Gruberstrasse 40, 4020 Linz, Austria.
authors: Christian
authors: C
authors: Rankl
authors: Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and.
authors: Lai-Xi
authors: LX
authors: Wang
authors: From the Institute for Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria.
authors: Hermann J
authors: HJ
authors: Gruber
authors: Max Planck Institute of Molecular Physiology, Otto-Hahn Strasse 11, 44227 Dortmund, Germany.
authors: Rolf K H
authors: RK
authors: Kinne
authors: From the Institute for Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz, Austria, peter.hinterdorfer@jku.at.
authors: Peter
authors: P
authors: Hinterdorfer
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D051269:Sodium-Glucose Transporter 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051269:Sodium-Glucose Transporter 1
keywords: Atomic Force Microscopy (AFM); Glucose Translocation Pathway; Glucose Transport; Kinetics; Ligand-binding Protein; Off Rate Constant; Phlorizin Binding; SGLT1; Spectroscopy; Width of Energy Barrier