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

Abstract

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.

Current problems and future avenues in proteoliposome research

“…Other approaches for guided orientation employed Ni-NTA-functionalized beads to immobilize His-tagged MPs prior to reconstitution, [96][97][98] a method also used to form planar bilayers for AFM studies. 99,100 The liposomal membrane was formed de novo between the immobilized MPs either around the bead-support, 96 or the beads were suggested to force unidirectional orientation because they were too big to be incorporated into the newly formed liposomes. 97,98 However, a more thorough characterization of these methods, e.g.…”

Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins

“…Due to their two-dimensional structure, SLBs are well suited for surface-sensitive applications, such as biochips (Chadli et al, 2018) and biosensors (Rebaud et al, 2014). Further, they are highly amenable to analysis by quartz crystal microbalance (Meléndrez et al, 2018), AFM (Bronder et al, 2016), X-ray (Kuhl et al, 2009; Xu et al, 2018), and neutron (Lind et al, 2015) scattering. Although conventional SLBs are formed on hard substrates (e.g., glass), an SLB can alternatively be modified to incorporate a polymer layer (El-khouri et al, 2011; Watkins et al, 2011; Pace et al, 2015; Andersson and Köper, 2016) between the bilayer and underlying hard substrate.…”

Tethered Membrane Architectures—Design and Applications

“…AFM studies of membrane proteins in a native conformation can provide valuable insight into their structure and function, but they are challenging to carry out. Using an architecture comprised of tris-NTA (to which the protein can bind trough a His-tag) and anchorlipid bound to the solid support via a PEGchain, an environment was created that enabled the study of bacteriorhodopsin as a model for membrane proteins (Bronder et al, 2016).…”