Conformation of Receptor Adopted upon Interaction with Virus Revealed by Site-Specific Fluorescence Quenchers and FRET Analysis

• Published in: Journal of the American Chemical Society 2009-04, Vol.131 (15), p.5478-5482
• Main Authors: Wruss, Jürgen, Pollheimer, Philipp D, Meindl, Irene, Reichel, Annett, Schulze, Katrin, Schöfberger, Wolfgang, Piehler, Jacob, Tampé, Robert, Blaas, Dieter, Gruber, Hermann J
• Format: Article
• Language: English
• Subjects: Binding Sites; Fluorescence; Fluorescence Resonance Energy Transfer; Genetic Engineering; Humans; Molecular Probe Techniques; Protein Binding; Protein Conformation; Receptors, LDL - metabolism; Rhinovirus - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja807917t

Human rhinovirus serotype 2 (HRV2) specifically binds to very-low-density lipoprotein receptor (VLDLR). Among the eight extracellular repeats of VLDLR, the third module (V3) has the highest affinity for the virus, and 12 copies of the genetically engineered concatamer V33333-His6 were found to bind per virus particle. In the present study, ring formation of V33333-His6 about each of the 12 5-fold symmetry axes on HRV2 was demonstrated by fluorescence resonance energy transfer (FRET) between donor and acceptor on N- and C-terminus, respectively. In particular, the N-terminus of V33333-His6 was labeled with fluorescein, and the C-terminus with a new quencher which was bound to the His6 tag with nanomolar affinity (K d ∼10−8 M) in the presence of 2 μM NiCl2.