title: Single-molecule force spectroscopy of membrane proteins from membranes freely spanning across nanoscopic pores.
abstract: Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.
authors: ‡Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, 4058 Basel, Switzerland.
authors: Rafayel
authors: R
authors: Petrosyan
authors: ‡Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, 4058 Basel, Switzerland.
authors: Christian A
authors: CA
authors: Bippes
authors: †Institute of Applied Physics and Center for Functional Nanostructures (CFN) and Institute of Nanotechnology (INT), Karlsruhe Institute of Technology (KIT), 76021 Karlsruhe, Germany.
authors: Stefan
authors: S
authors: Walheim
authors: §Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland.
authors: Daniel
authors: D
authors: Harder
authors: §Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland.
authors: Dimitrios
authors: D
authors: Fotiadis
authors: †Institute of Applied Physics and Center for Functional Nanostructures (CFN) and Institute of Nanotechnology (INT), Karlsruhe Institute of Technology (KIT), 76021 Karlsruhe, Germany.
authors: Thomas
authors: T
authors: Schimmel
authors: ‡Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, 4058 Basel, Switzerland.
authors: David
authors: D
authors: Alsteens
authors: ‡Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, 4058 Basel, Switzerland.
authors: Daniel J
authors: DJ
authors: Müller
mesh_terms: D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D018625:Microscopy, Atomic Force; D058608:Nanopores / Q000648:ultrastructure*; D036103:Nanotechnology; D013057:Spectrum Analysis
chemical_list: D008565:Membrane Proteins
keywords: AFM; atomic force microscopy; freely spanning membrane; molecular interactions; polymer blend lithography; unfolding intermediates