Ian L Martin | PubMed Health

PubMed2005

Atomic force microscopy reveals the stoichiometry and subunit arrangement of 5-HT3 receptors.

The 5-HT3 receptor is a cation-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor is an important therapeutic target, with receptor antagonists being widely used as antiemetics in cancer therapy. The two known receptor subunits, A and B, form homomeric 5-HT 3A receptors and heteromeric 5-HT 3A/B receptors. The heteromeric receptor has the higher single-channel conductance and more closely mimics the properties of the native receptor. We have used atomic force microscopy to study the architecture of 5-HT 3A and 5-HT 3A/B receptors. We engineered different epitope tags onto the A- and B-subunits and imaged receptors that were doubly liganded by anti-epitope antibodies. We found that, for the 5-HT 3A/B receptor, the distribution of angles between antibodies against the A-subunit had a single peak at approximately 144 degrees , whereas the distribution for antibodies against the B-subunit had two peaks at approximately 72 degrees and 144 degrees . Our results indicate that the subunit stoichiometry is 2A:3B and that the subunit arrangement around the receptor rosette is B-B-A-B-A. This arrangement may account for the difference between the agonist Hill coefficients and the single-channel conductances for the two types of receptor.

Animals↗

PubMed2002

Direct visualization of ligand-protein interactions using atomic force microscopy.

1. Streptavidin is a 60-kDa tetramer which binds four molecules of biotin with extremely high affinity (K(A) approximately 10(14) M(-1)). We have used atomic force microscopy (AFM) to visualize this ligand-protein 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 beta-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 effect 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.

Binding Sites↗