High-Resolution Imaging and Multiparametric Characterization of Native Membranes by Combining Confocal Microscopy and an Atomic Force Microscopy-Based Toolbox.

Laskowski, Pawel R; Pfreundschuh, Moritz; Stauffer, Mirko; Ucurum Fotiadis, Zöhre; Fotiadis, Dimitrios José; Müller, Daniel J (2017). High-Resolution Imaging and Multiparametric Characterization of Native Membranes by Combining Confocal Microscopy and an Atomic Force Microscopy-Based Toolbox. ACS nano, 11(8), pp. 8292-8301. American Chemical Society 10.1021/acsnano.7b03456

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Stauffer, Mirko; Ucurum Fotiadis, Zöhre and Fotiadis, Dimitrios José

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

1936-0851

Publisher:

American Chemical Society

Language:

English

Submitter:

Barbara Järmann-Bangerter

Date Deposited:

15 Mar 2018 16:16

Last Modified:

15 Mar 2018 16:16

Publisher DOI:

10.1021/acsnano.7b03456

PubMed ID:

28745869

Uncontrolled Keywords:

chemical recognition imaging fluorescence microscopy force spectroscopy ligand binding membrane protein multiparametric imaging single-molecule imaging

BORIS DOI:

10.7892/boris.110076

URI:

https://boris.unibe.ch/id/eprint/110076

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