力谱学
嗜盐菌
膜
细菌视紫红质
生物物理学
膜蛋白
膜生物学
显微镜
化学
原子力显微镜
共焦显微镜
纳米技术
材料科学
生物
生物化学
细胞生物学
光学
物理
作者
Pawel R. Laskowski,Moritz Pfreundschuh,Mirko Stauffer,Zöhre Ucurum,Dimitrios Fotiadis,Daniel J. Müller
出处
期刊:ACS Nano
[American Chemical Society]
日期:2017-08-01
卷期号:11 (8): 8292-8301
被引量:23
标识
DOI:10.1021/acsnano.7b03456
摘要
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.
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