Single molecule recognition of protein binding epitopes in brush border membranes by force microscopy.
Sidedness and accessibility of protein epitopes in intact brush border membrane vesicles were analyzed by detecting single molecule interaction forces using molecular recognition force microscopy in aqueous physiological solutions. Frequent antibody-antigen recognition events were observed with a force microscopy tip carrying an antibody directed against the periplasmically located gamma-glutamyltrans- peptidase, suggesting a right side out orientation of the vesicles. Phlorizin attached to the tips bound to NA+/D-glucose cotransporter molecules present in the vesicles. The recognition was sodium dependent and inhibited by free phlorizin and D-glucose, and revealed an apparent K(D) of 0.2 microM. Binding events were also observed with an antibody directed against the epitope aa603-aa630 close to the C terminus of the transporter. In the presence of phlorizin the probability of antibody binding was reduced but the most probable unbinding force f(u) = 100 pN remained unchanged. In the presence of D-glucose and sodium, however, both the binding probability and the most probable binding force (f(u) = 50 pN) were lower than in its absence. These studies demonstrate that molecular recognition force microscopy is a versatile tool to probe orientation and conformational changes of epitopes of membrane components during binding and trans-membrane transport.
Wielert-Badt
Susanne
S
Max-Planck-Institute for Molecular Physiology, Department of Epithelial Cell Physiology, 44227 Dortmund, Germany.
Hinterdorfer
Peter
P
Gruber
Hermann J
HJ
Lin
Jiann-Trzuo
JT
Badt
Dirk
D
Wimmer
Barbara
B
Schindler
Hansgeorg
H
Kinne
Rolf K-H
RK
D000818:Animals; D000906:Antibodies; D001666:Binding Sites, Antibody; D000939:Epitopes / Q000032:analysis*; D007202:Indicators and Reagents; D007672:Kidney Cortex / Q000737:chemistry / Q000648:ultrastructure; D008024:Ligands; D018625:Microscopy, Atomic Force / Q000379:methods; D008871:Microvilli / Q000737:chemistry* / Q000648:ultrastructure; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000276:immunology; D010695:Phlorhizin; D011506:Proteins / Q000032:analysis*; D011817:Rabbits
D000906:Antibodies; D000939:Epitopes; D007202:Indicators and Reagents; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D011506:Proteins; D010695:Phlorhizin