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J Mol Recognit ; 28(1): 1-9, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26046654

RESUMEN

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the ß2-adrenergic receptor (ß2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of ß2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process.


Asunto(s)
Membrana Celular/metabolismo , Pared Celular/metabolismo , Glicoproteínas de Membrana/química , Microscopía de Fuerza Atómica/métodos , Proteínas de Saccharomyces cerevisiae/química , Animales , Células CHO , Línea Celular , Cricetulus , Proteínas Fúngicas/química , Proteínas Fúngicas/inmunología , Proteínas Fúngicas/metabolismo , Hemaglutininas/química , Hemaglutininas/inmunología , Hemaglutininas/metabolismo , Humanos , Gripe Humana/metabolismo , Glicoproteínas de Membrana/metabolismo , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Receptores Adrenérgicos/química , Receptores Adrenérgicos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
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