1 Streptavidin is a 60-kDa tetramer which binds four molecules of biotin with extremely high anity (K A *10 14 M 71). We have used atomic force microscopy (AFM) to visualize this ligandprotein interaction directly. 2 Biotin was tagged with a short (152-basepair; 50-nm) DNA rod and incubated with streptavidin. The resulting complexes were then imaged by AFM. The molecular volume of streptavidin calculated from the dimensions of the protein particles (105+3 nm 3 ) was in close agreement with the value calculated from its molecular mass (114 nm 3 ). Biotinylation increased the apparent size of streptavidin (to 133+2 nm 3 ), concomitant with an increase in the thermal stability of the tetramer. 3 Images of streptavidin with one to four molecules of DNA-biotin bound were obtained. When two ligands were bound, the angle between the DNA rods was either acute or obtuse, as expected from the relative orientations of the biotin binding sites. The ratio of acute : obtuse angles (1 : 3) was lower than the expected value (1 : 2), indicating a degree of steric hindrance in the binding of the DNA-biotin. The slight under-representation of higher occupancy states supported this idea. 4 Streptavidin with a single molecule of DNA-biotin bound was used to tag biotinylated b-galactosidase, a model multimeric enzyme. 5 The ability to image directly the binding of a ligand to its protein target by AFM provides useful information about the nature of the interaction, and about the eect of complex formation on the structure of the protein. Furthermore, the use of DNA-biotin/streptavidin tags could potentially shed light on the architecture of multi-subunit proteins.

We have developed an atomic force microscopy (AFM)-based method for the determination of the subunit architecture of ionotropic receptors, and tested the method using the GABAA receptor as a model system. The most common form of the GABAA receptor probably consists of 2α1-, 2β2- and 1γ2-subunits. We show here that the arrangement of subunits around the central Cl− ion channel can be deduced by AFM of receptors tagged with subunit-specific antibodies. Transfection of cells with DNA encoding α1-, β2- and γ2-subunits resulted in the production of receptors containing all three subunits, as judged by both immunoblot analysis and the binding of [3H]-Ro15-1788, a specific radioligand for the GABAA receptor. A His6-tag on the α1-subunit was used to purify the receptor from membrane fractions of transfected cells. After incubation with anti-His6 immunoglobulin G, some receptors became tagged with either one or two antibody molecules. AFM analysis of complexes containing two bound antibodies showed that the most common angle between the two tags was 135°, close to the value of 144° expected if the two α-subunits are separated by a third subunit. This method is applicable to the complete elucidation of the subunit arrangement around the GABAA receptor rosette, and can also be applied to other ionotropic receptors.