Single-Molecule Force Spectroscopy Measures Structural Changes Induced by Light Activation and Transducer Binding in Sensory Rhodopsin II

Oberbarnscheidt, Leoni; Janissen, Richard; Martell, Swetlana; Engelhard, Martin; Oesterhelt, Filipp

doi:10.1016/j.jmb.2009.07.083

Cited by 4 publications

(4 citation statements)

References 39 publications

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“…69,91,92 Furthermore, SMFS was shown to be sufficiently sensitive to detect and localize interactions that induce conformational changes of membrane proteins. 33,38,93,94 Thus, these SMFS results obtained on b 2 AR suggest that ligand binding does not introduce strong localized interactions to change the functional state of the GPCR. Similarly, SMFS on bovine rhodopsin in the ligand bound dark state and opsin in the ligand free activated state revealed no changes of the force peaks.…”

Section: Characterizing the Energy Landscape Of Gpcrs Changing Upon L...mentioning

confidence: 66%

Single-molecule force spectroscopy of G-protein-coupled receptors

et al. 2013

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The applicability of single-molecule force spectroscopy (SMFS) to characterize membrane proteins in vitro is developing rapidly and opening a wide range of fascinating possibilities to study how intra- and intermolecular interactions determine their structural stability and functional state. In particular, understanding how molecular interactions contribute to the functional state of G-protein-coupled receptors (GPCRs) is of importance because they mediate most of our physiological responses and act as therapeutic targets for a broad spectrum of diseases. In our review we focus on SMFS to characterize GPCRs embedded in their physiologically relevant membranes and exposed to physiologically relevant conditions. SMFS uses a molecularly sharp stylus to grasp the terminal end of a GPCR and to quickly unfold the receptor while recording interaction forces. The positional accuracy of SMFS localizes these interactions to structural segments of the GPCR whereas the sensitivity of SMFS enables their stabilizing interaction forces to be quantified. To further investigate the kinetic, energetic and mechanical properties of the structural segments, dynamic SMFS (DFS) probes their stability over a wide range of loading rates. These parameters provide insight into the energy landscape that provides information on the structural and functional properties of the GPCRs. Selected highlights exemplify the application of SMFS to characterize inter- and intramolecular interactions, which change the properties of GPCRs in relation to their functional state (e.g., ligand binding), diseased state (e.g., mutation), or lipid environment such as cholesterol.

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Section: Characterizing the Energy Landscape Of Gpcrs Changing Upon L...mentioning

confidence: 66%

Single-molecule force spectroscopy of G-protein-coupled receptors

et al. 2013

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“…Proteins embedded in a tBLM can be studied by atomic force microscopy (AFM). AFM-based single-molecule force spectroscopy can be used to obtain information on dissociation rates (19), energy barriers (20,21), Gibbs free energy (19,21), the form of a binding potential (21), and inter-and intramolecular interactions (20,(22)(23)(24), as well as the folding of proteins and their constitution inside a membrane (20).…”

Section: Introductionmentioning

confidence: 99%

Oriented Membrane Protein Reconstitution into Tethered Lipid Membranes for AFM Force Spectroscopy

Bronder

Bieker

Elter

et al. 2016

Biophysical Journal

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Membrane proteins act as a central interface between the extracellular environment and the intracellular response and as such represent one of the most important classes of drug targets. The characterization of the molecular properties of integral membrane proteins, such as topology and interdomain interaction, is key to a fundamental understanding of their function. Atomic force microscopy (AFM) and force spectroscopy have the intrinsic capabilities of investigating these properties in a near-native setting. However, atomic force spectroscopy of membrane proteins is traditionally carried out in a crystalline setup. Alternatively, model membrane systems, such as tethered bilayer membranes, have been developed for surface-dependent techniques. While these setups can provide a more native environment, data analysis may be complicated by the normally found statistical orientation of the reconstituted protein in the model membrane. We have developed a model membrane system that enables the study of membrane proteins in a defined orientation by single-molecule force spectroscopy. Our approach is demonstrated using cell-free expressed bacteriorhodopsin coupled to a quartz glass surface in a defined orientation through a protein anchor and reconstituted inside an artificial membrane system. This approach offers an effective way to study membrane proteins in a planar lipid bilayer. It can be easily transferred to all membrane proteins that possess a suitable tag and can be reconstituted into a lipid bilayer. In this respect, we anticipate that this technique may contribute important information on structure, topology, and intra-and intermolecular interactions of other seven-transmembrane helical receptors.

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“…In our group we have studied bacterial membrane proteins, e.g. bacteriorhodopsin, with single-molecule atomic force microscopy and spectroscopy [4,5]. The atomic force microscope (AFM) is a tool to image biological surfaces with sub-nanometer resolution.…”

Section: Introductionmentioning

confidence: 99%

“…Due to this it was already possible to show new locations for structural changes in sensory rhodopsin 2 upon light activation. It was also demonstrated, that the conformational answer after light activation varies if another protein is bound to sensory rhodopsin 2 [4]. Further studies on the effect of compatible solutes on bacteriorhodopsin showed a general stabilization of membrane proteins by ectoine [5].…”

Section: Introductionmentioning

confidence: 99%