High-Resolution Imaging and Multiparametric Characterization of Native Membranes by Combining Confocal Microscopy and an Atomic Force Microscopy-Based Toolbox Pawel R. Laskowski,† Moritz Pfreundschuh,† Mirko Stauffer,‡ Zöhre Ucurum,‡ Dimitrios Fotiadis,‡ and Daniel J. Müller*,† †
Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland
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S Supporting Information *
ABSTRACT: To understand how membrane proteins function requires characterizing their structure, assembly, and inter- and intramolecular interactions in physiologically relevant conditions. Conventionally, such multiparametric insight is revealed by applying different biophysical methods. Here we introduce the combination of confocal microscopy, force−distance curve-based (FD-based) atomic force microscopy (AFM), and single-molecule force spectroscopy (SMFS) for the identification of native membranes and the subsequent multiparametric analysis of their membrane proteins. As a well-studied model system, we use native purple membrane from Halobacterium salinarum, whose membrane protein bacteriorhodopsin was His-tagged to bind nitrilotriacetate (NTA) ligands. First, by confocal microscopy we localize the extracellular and cytoplasmic surfaces of purple membrane. Then, we apply AFM to image single bacteriorhodopsins approaching sub-nanometer resolution. Afterwards, the binding of NTA ligands to bacteriorhodopsins is localized and quantified by FD-based AFM. Finally, we apply AFM-based SMFS to characterize the (un)folding of the membrane protein and to structurally map inter- and intramolecular interactions. The multimethodological approach is generally applicable to characterize biological membranes and membrane proteins at physiologically relevant conditions. KEYWORDS: chemical recognition imaging, fluorescence microscopy, force spectroscopy, ligand binding, membrane protein, multiparametric imaging, single-molecule imaging
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individual method and to provide multiparametric information on biological membranes with increasing efficiency.3,6,9−11 Confocal microscopy and AFM are complementary. While fluorescence microscopy is routinely applied in life sciences to characterize fluorescently labeled samples, confocal fluorescence microscopy can be used to image complex biological objects in three dimensions. Conventionally, confocal microscopy can approach a vertical resolution of ∼500−800 nm and a lateral resolution of ∼200−500 nm. Invented more than 30 years ago, AFM enables contouring solid-state samples to atomic resolution.12−14 The combination of high-resolution AFM imaging with the possibility to use the AFM tip to
ellular membranes dynamically reassemble membrane proteins and lipids in order to guide their function.1,2 Depending on this heterogeneous assembly, inter- and intramolecular interactions change, thereby modulating the functional state of membrane proteins. Hence, high-resolution imaging of membrane proteins in the native membrane and at physiologically relevant conditions along with quantifying their physical and chemical characteristics is necessary for the comprehensive understanding of how these proteins work and are regulated. A growing number of methods suitable for exploring the biophysical and biochemical properties of biological membranes and membrane proteins at high resolution, including optical microscopy and spectroscopy, electron microscopy, and atomic force microscopy (AFM), has increased interest in the field.3−9 However, optimized approaches are necessary to mitigate the shortcomings of any © 2017 American Chemical Society
Received: May 17, 2017 Accepted: July 26, 2017 Published: July 26, 2017 8292
DOI: 10.1021/acsnano.7b03456 ACS Nano 2017, 11, 8292−8301
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Figure 1. Combined confocal fluorescence microscopy and FD-based AFM imaging. (a) Experimental setup. A thin layer of mica (
