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.
Neish
Calum S
CS
Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK.
Martin
Ian L
IL
Henderson
Robert M
RM
Edwardson
J Michael
JM
D001665:Binding Sites / Q000502:physiology; D001710:Biotin / Q000378:metabolism; D008024:Ligands; D018625:Microscopy, Atomic Force / Q000379:methods*; D011506:Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D019809:Streptavidin / Q000378:metabolism / Q000648:ultrastructure; D001616:beta-Galactosidase / Q000378:metabolism / Q000648:ultrastructure
D008024:Ligands; D011506:Proteins; D001710:Biotin; D019809:Streptavidin; D001616:beta-Galactosidase