Biological membranes compartmentalize and define physical borders of cells. They are crowded with membrane proteins that fulfill diverse crucial functions. About one-third of all genes in organisms code for, and the majority of drugs target, membrane proteins. To combine structure and function analysis of membrane proteins, we designed a two-chamber atomic force microscopy (AFM) setup that allows investigation of membranes spanned over nanowells, therefore separating two aqueous chambers. We imaged nonsupported surface layers (S layers) of Corynebacterium glutamicum at sufficient resolution to delineate a 15 A-wide protein pore. We probed the elastic and yield moduli of nonsupported membranes, giving access to the lateral interaction energy between proteins. We combined AFM and fluorescence microscopy to demonstrate the functionality of proteins in the setup by documenting proton pumping by Halobacterium salinarium purple membranes.
The Voltage Dependent Anion Channel (VDAC) is the most abundant protein in the outer membrane of mitochondria. This strategic localization puts it at the heart of a great number of phenomena. Its recent implication in apoptosis is an example of the major importance of this protein and has created a surge of interest in VDAC. There is no atomic-resolution structure allowing a better understanding of the function of VDAC, so alternative techniques to X-ray diffraction have been used to study VDAC. Here we discuss structural models from folding predictions and review data acquired by Atomic Force Microscopy (AFM) imaging that allowed to observe VDAC's structure and supramolecular organization in the mitochondrial outer membrane.
Membrane proteins are nanometric machines fulfilling defined functions in the membranes of all living cells. They work as transporters, linkers, adhesion molecules, channels, pumps, receptors and enzymes, and in bio-energetic machineries, to name only a few tasks. In agreement with their multiple functions and importance, it was found that about 25% of all genes code for membrane proteins in organisms ranging from bacteria to humans. Biologists now have a set of techniques such as X-ray crystallography, electron microscopy, and atomic force microscopy to analyze membrane protein structure, and techniques such as patch-clamp, black lipid membrane and spectroscopy on membrane vesicles to analyze membrane function. Atomic force microscopy is widely used for imaging and force measurements, and here, its use as a unique tool to nano-manipulate individual membrane proteins is reviewed. In this 'mode', additional loading forces are applied to an imaging tip, and scan rates and feedback parameters are adjusted to deliberately act on the surface of the biological object. When additional loading forces are relatively high (∼500 pN), stacked membrane layers can be dissected to give access to underlying membranes. Similarly, subunits from a multi-protein complex can be dissected at slightly increased forces (∼200 pN) allowing the analysis of underlying protein structures, and hence that of the complex architecture. At low additional loading forces (∼100 pN), individual protein loops can be manipulated. Importantly, this process is nondestructive and provides access for the analysis of flexible protein surface domains.
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