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Direct Observation of Ligand Binding to Membrane Proteins in Living Cells by a Saturation Transfer Double Difference (STDD) NMR Spectroscopy Method Shows a Significantly Higher Affinity of Integrin α IIb β 3 in Native Platelets than in Liposomes

2004; American Chemical Society; Volume: 127; Issue: 3 Linguagem: Inglês

10.1021/ja044434w

1943-2984

Birgit Claasen, Marco Axmann, Robert Meinecke, Bernd Meyer,

Platelet Disorders and Treatments

About 30% of the proteins in mammalian systems are membrane bound or integrated (e.g., GPCRs). It is inherently difficult to investigate receptor−ligand interactions on a molecular level in their natural membrane environment. Here, we present a new method based on saturation transfer difference (STD) NMR to characterize at an atomic level binding interactions of cell surface proteins in living cells. Implemented as a double difference technique, STD NMR allows the direct observation of binding events and the definition of the binding epitopes of ligands. The binding of the pentapeptide cyclo(RGDfV) to the surface glycoprotein integrin αIIbβ3 of intact human blood platelets can be detected by saturation transfer double difference (STDD) NMR in less than an hour. A 5-fold higher STD response reflects a significantly higher affinity of integrin αIIbβ3 in native platelets than in liposomes, which demonstrates the importance of studying membrane proteins in their natural environment. Also, the binding mode of cyclo(RGDfV) in the arginine glycine region is slightly different when interacting with native integrin in platelets compared to integrin reintegrated into liposomes.