Manipulating and imaging individual membrane proteins by AFM

Abstract

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.

Native architecture of the photosynthetic membrane from Rhodobacter veldkampii

“…In spite of the structural information of individual photosynthetic components, knowledge of the macromolecular organization of these protein complexes in the native state required for understanding the physiological activities and functional cooperativity of the photosynthetic apparatus awaited the dawn of atomic force microscopy (AFM). AFM with high lateral resolution and high signal-to-noise ratio has evolved into a powerful tool to directly and precisely visualize biological samples under physiological conditions (Engel and Gaub, 2008;Gonçalves and Scheuring, 2006;Scheuring, 2006). To date, AFM has significantly advanced the elucidation of the native surface views of ICMs from different photosynthetic bacteria, including lamellar discs in Blastochloris (Blc.)…”

Making the Most of Fusion Tags Technology in Structural Characterization of Membrane Proteins

“…For example, Barrera et al [18] determined the subunit stoichiometry of the transient receptor potential C1 (TRPC1) channel, fused with affinity tags and epitope tags, by imaging isolated channels with AFM and monoclonal antibodies. AFM gives a simple, rapid and convenient way to image the shape of protein specimens [37]. By measuring and calculating the molecular volumes of individual channel particles, they found that TRPC1 exists both in monomeric and tetrameric forms.…”

“…The high-resolution three-dimensional structure of membrane proteins can be elucidated by means of NMR spectroscopy [34], X-ray crystallography [35], cryo-electron microscopy [36], or atomic force microscopy (AFM) [37]. Although nearly one third of all open reading frames in fully sequenced genomes are predicted to encode membrane proteins [1,2], it is extremely difficult to obtain high-resolution three-dimensional structures of membrane proteins.…”

Quinone Pathways in Entire Photosynthetic Chromatophores of Rhodospirillum photometricum

“…10,14,15 Based on the advantages of the technique, AFM has significantly advanced the elucidation of the native architecture of photosynthetic membranes. The first views of the photosynthetic apparatus at submolecular resolution provided insights into the multicomponent energy-transducing membranes from different photosynthetic bacteria: Blastochloris viridis, 16 Rhodospirillum photometricum, [17][18][19] Rhodobacter sphaeroides, 20 Rhodobacter blasticus, 21 Phaeospirillum molischianum 22 and Rhodopseudomonas palustris.…”