RESUMEN
The Rh blood group system plays a key role in transfusion and organ transplant medicine. The complex transmembrane Rh polypeptides RhD and RhCE carry numerous antigens, including the extremely immunogenic D antigen. The Rh polypeptides form multimolecular Rh complexes with certain transmembrane and skeletal proteins, with so far only incompletely understood physiological functions. Determination of the energy landscape of individual Rh binding epitopes towards their specific interaction partners as well as their localization across the red blood cell (RBC) membrane requires single molecule approaches including large area high resolution recognition imaging. Atomic force microscopy based molecular recognition force spectroscopy in combination with single molecule recognition imaging fulfills these requirements. For unbiased single molecule results, nano-mechanical influences due to cell elasticity have to be eliminated. This is realized by generation of ultra flat erythrocyte ghosts on a solid support. We developed a protocol for the preparation of complete ultraflat erythrocyte ghosts and determined the molecular binding behaviour of different anti-D antibodies towards their binding epitopes on RhD positive and negative erythrocytes. Performing optimized topography and recognition imaging at 16 Mpixel resolution allowed localisation of individual RhD molecules at the single molecule level across an entire RBC. A map of Rh antigens across integer ultraflat RBC ghosts was generated with nanometer resolution. Here we show a homogeneous distribution on rim and dimple regions with comparable receptor densities. Furthermore, differences in the energy landscape between specific monoclonal antibodies were determined at the single molecule level.
Asunto(s)
Membrana Eritrocítica , Eritrocitos , Anticuerpos Monoclonales , EpítoposRESUMEN
Crystal structures and experiments relying on the tools of molecular pharmacology reported conflicting results on ligand binding sites in neurotransmitter/sodium symporters (NSS). We explored the number and functionality of ligand binding sites of NSS in a physiological setting by designing novel tools for atomic force microscopy (AFM). These allow for directly measuring the interaction forces between the serotonin transporter (SERT) and the antidepressant S-citalopram (S-CIT) on the single molecule level: the AFM cantilever tips were functionalized with S-CIT via a flexible polyethylene glycol (PEG) linker. The tip chemistry was validated by specific force measurements and recognition imaging on CHO cells. Two distinct populations of characteristic binding strengths of S-CIT binding to SERT were revealed in Na+-containing buffer. In contrast, in Li+-containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT. The dissociation rate constant of both binding sites was extracted by varying the dynamics of the force-probing experiments. Competition experiments revealed that the two sites are allosterically coupled and exert reciprocal modulation.