Oriented Membrane Protein Reconstitution into Tethered Lipid Membranes for AFM Force Spectroscopy.
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.
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany. Electronic address: anna.bronder@hhu.de.
Anna M
AM
Bronder
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Adeline
A
Bieker
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Shantha
S
Elter
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Manuel
M
Etzkorn
Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-Universität, Düsseldorf, Germany.
Dieter
D
Häussinger
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany; Department for Microbial Bioactive Compounds, Interfaculty Institute for Microbiology and Infection Medicine, University of Tuebingen, Tuebingen, Germany.
Filipp
F
Oesterhelt
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D008567:Membranes, Artificial; D018625:Microscopy, Atomic Force; D013499:Surface Properties
D008567:Membranes, Artificial; D001436:Bacteriorhodopsins