Manipulating and imaging individual membrane proteins by AFM

摘要

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 (similar to 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 (similar to 200 pN) allowing the analysis of underlying protein structures, and hence that of the complex architecture. At low additional loading forces (similar to 100 pN), individual protein loops can be manipulated. Importantly, this process is nondestructive and provides access for the analysis of flexible protein surface domains. Copyright (C) 2006 John Wiley & Sons, Ltd.