Biophysical methods to study ligand-receptor interactions of neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acids peptide amide that was isolated for the first time almost 20 years ago from porcine brain. NPY displays a multiplicity of physiological effects that are transmitted by at least six G-protein coupled receptors (GPCRs) named Y(1), Y(2), Y(3), Y(4), Y(5), and y(6). Because of the difficulty in obtaining high-resolution crystallographic structures from GPCRs that all belong to seven transmembrane helices proteins, a variety of biophysical methods have been applied in order to characterize the interaction of ligand and receptor. In this review article we present the most relevant outcomes of the studies performed in this field by our group and others. The use of photoaffinity labeling allowed the molecular characterization of the Y(2) receptor. The concerted application of molecular modeling and mutagenesis studies led to a model for the interaction of the natural agonist and nonpeptide antagonists with the Y(1) receptor. The three-dimensional (3D) structure and dynamics of micelle-bound NPY and their implications for receptor selection have been studied by NMR. The characterization of the tertiary and quaternary structure of the NPY dimer in solution at millimolar concentrations has been performed by NMR and extended to physiologically relevant concentrations by fluorescence resonance energy transfer (FRET) experiments performed with fluorescence-labeled analogues.