Single Molecule Recognition of Protein Binding Epitopes in Brush Border Membranes by Force Microscopy

Abstract

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.

Single-molecule recognition imaging microscopy

“…6, which is published as supporting information on the PNAS web site. The force rises rapidly above 20 pN for z͞L 0 Ͼ 0.5, leading to potentially increased frequency of breaking of a typical antibody-antigen bond (16). Near this optimum value of z͞L 0 , S(z͞L 0 ) is Ϸ2 pN͞nm.…”

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…Host-guest complexes have played a predominant role in the study of specific forces. Several experiments have been performed with different complementary molecules in the last years [368,[1118][1119][1120][1121][1122][1123][1124][1125][1126][1127][1128][1129]. It also has been shown, that force-displacement curves with functionalized tips can be employed to distinguish molecules of different chirality [1130,1131].…”

C-terminal Loop 13 of Na+/Glucose Cotransporter 1 Contains Both Stereospecific and Non-stereospecific Sugar Interaction Sites

“…Thus, discrimination between D-and L-glucose was concluded to be achieved by an initial, extracellularly occurring, binding event which precedes the intrinsic translocation across the membrane (10). Our group also provided evidence for an involvement of late loop 13 (aa 564 -638) in stereospecific sugar selectivity of rabbit SGLT1 as binding of PAN3-2 antibody to the whole cotransporter could be blocked by D-glucose but not by L-glucose during atomic force microscopy force distance cycles (4,8). PAN3-2 is directed against aa 606 -630 and, thus, reports mainly on changes in the late part of loop 13.…”

“…Evidence for such an orientation is provided by the presence of a phosphorylation consensus site, spanning aa 561-564 on the early loop (33), and the detection of the late part of loop 13 on the outer surface of SGLT1 expressing cells in single molecule recognition atomic force microscopy experiments (4,8).…”

“…Phlorizin, a ␤-glucoside of the aromatic compound phloretin, is the most potent inhibitor with an apparent K i value of 1 M (11). In previous studies we have shown that phlorizin binds to loop 13 of rabbit SGLT1 (aa 541-638) via the aromatic aglucone moiety inducing conformational changes of the loop, predominantly in the region between aa 600 and aa 621 (4,6) of the C terminus. Mutagenesis experiments provided also data on individual amino acids critically important for the interaction with the inhibitors (6,13,14).…”