Single-molecule studies of membrane proteins Daniel J Mu ¨ ller 1 , K Tanuj Sapra 1 , Simon Scheuring 2 , Alexej Kedrov 1 , Patrick L Frederix 3 , Dimitrios Fotiadis 3 and Andreas Engel 3 Characterizing membrane proteins with single-molecule techniques provides structural and functional insights that cannot be obtained with conventional approaches. Recent studies show that atomic force microscopy (AFM) in the context of a ‘lab on a tip’ enables the measurement of multiple parameters of membrane proteins. This multifunctional tool can be applied to probe the oligomeric states and conformational changes of membrane protein assemblies in their native environment. The ability to determine diverse properties at high spatial resolution facilitates the mapping of structural flexibilities, electrostatic potentials and electric currents. By using the AFM tip as tweezer, it is possible to characterize unfolding and refolding pathways of single proteins and the location of their molecular interactions. These interactions dictate the stability of the protein and might be modulated by ligands that alter the protein’s functional state. Addresses 1 Center for Biotechnology, University of Technology, 01307 Dresden, Germany 2 Institut Curie, UMR168-CNRS, 26 Rue d’Ulm, 75248 Paris, France 3 M.E. Mu ¨ ller Institute for Microscopy, Biozentrum, University of Basel, 4056 Basel, Switzerland Corresponding author: Mu ¨ ller, Daniel J (mueller@biotec.tu-dresden.de) Current Opinion in Structural Biology 2006, 16:489–495 This review comes from a themed issue on Membranes Edited by Roderick MacKinnon and Gunnar von Heijne Available online 23rd June 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.06.001 Introduction Biological membranes comprise dynamic associations of membrane proteins and lipids that adapt to functional requirements. A simple organism such as Escherichia coli hosts more than a thousand helical transmembrane pro- teins in its plasma membrane, giving more than half a million types of pairwise combinations. Regions of biased composition exist and the protein environments vary from time-invariant complexes and transient associations to biased distributions. New approaches are required to access this dynamic structure of cell membranes and to visualize membrane protein organization in vivo. Single-molecule techniques are used to study how mem- brane proteins interact and how they associate with each other to form higher order structures. Many of these approaches are based on light or electron microscopy. In contrast to these methods, atomic force microscopy (AFM) exhibits a superior signal-to-noise ratio that enables every single protein in the membrane to be observed. AFM does not require labeling of proteins, but images them in buffer solution at ambient tempera- tures even in densely packed assemblies found in native membrane patches. Here, an overview is given of the recent progress in the use of AFM for studying mem- branes. By using a multifunctional ‘lab on a tip’ device, various parameters of membrane proteins in their native environment can be characterized. We summarize recent findings on oligomeric protein assemblies, protein asso- ciations in the native membrane, folding and unfolding pathways of single membrane proteins and the detection and location of ligand binding. AFM: a lab on a tip The heart of the AFM is a several tens of micrometer long cantilever with a probe mounted at its end (Figures 1a and 1b). In the imaging mode, the probe raster scans the surface of a membrane, thereby contouring its profile. The best resolution achieved vertically is 0.1 nm and laterally 0.5 nm, enabling structural details of single membrane proteins to be observed. Structural comparison of the AFM topographs with atomic structures obtained by X-ray and electron crystallography demonstrates that protein structures are not disturbed by the imaging pro- cess. Moreover, the analysis of single membrane protein images shows (Figure 1d) that some structural regions exhibit intrinsic flexibilities and adopt particular confor- mations [1,2]. Sampling the conformational states of these structural regions enables the determination of a variety of parameters that describe the mechanical properties of a membrane protein surface. Minor structural fluctuations reflect the rigid parts of the molecule and large vertical variations the flexible parts. For example, vertical fluctua- tions can emerge from lateral displacements of polypep- tide loops connecting transmembrane a-helices. Calculating the probabilities for different structural regions to exist in a distinct conformation provides a map that can be readily converted to a free energy land- scape [2]. This map indicates the structurally flexible regions that undergo conformational changes [3,4]. In the future, such approaches will be used to investigate how environmental changes or ligand binding affects the energy landscape [5,6] of native protein surfaces. Based on its chemical characteristics and mechanical design, the AFM probe interacts differently with the www.sciencedirect.com Current Opinion in Structural Biology 2006, 16:489–495