Imaging the spatial orientation of subunits within membrane receptors by atomic force microscopy.

Our experimental approach is based on the atomic force microscope (AFM) imaging of epitope-tagged subunits within membrane protein complexes purified in small amounts and decorated by anti-tag antibodies. Furthermore, we can produce simultaneous decoration of protein complexes using Fab fragments and IgG antibodies, which, combined with chemical modification of the substrate, allows us to determine the protein orientation across the cell membrane. Here, we describe a detailed protocol for membrane protein purification, AFM data collection, analysis, and interpretation of results.

Structure and binding mechanism of vascular endothelial cadherin: a divergent classical cadherin.

Vascular endothelial cadherin (VE-cadherin), a divergent member of the type II classical cadherin family of cell adhesion proteins, mediates homophilic adhesion in the vascular endothelium. Previous investigations with a bacterially produced protein suggested that VE-cadherin forms cell surface trimers that bind between apposed cells to form hexamers. Here we report studies of mammalian-produced VE-cadherin ectodomains suggesting that, like other classical cadherins, VE-cadherin forms adhesive trans dimers between monomers located on opposing cell surfaces.

Acid-sensing ion channel (ASIC) 1a undergoes a height transition in response to acidification.

The acid-sensing ion channel (ASIC) 1a is known to assemble as a homotrimer. Here, we used atomic force microscopy to image ASIC1a, integrated into lipid bilayers, at pH 7.0 and pH 6.

Demonstration of a direct interaction between sigma-1 receptors and acid-sensing ion channels.

The sigma-1 receptor is a widely expressed protein that interacts with a variety of ion channels, including the acid-sensing ion channel (ASIC) 1a. Here we used atomic force microscopy to determine the architecture of the ASIC1a/sigma-1 receptor complex. When isolated His(8)-tagged ASIC1a was imaged in complex with anti-His(6) antibodies, the angle between pairs of bound antibodies was 135 degrees , consistent with the known trimeric structure of the channel.

Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping.

Atomic force microscopy (AFM) allows the study of single protein-DNA interactions such as those observed with the Type I Restriction-Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation.

Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging.

There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica.