High resolution nuclear magnetic resonance spectroscopy‐guided mutagenesis for characterization of membrane‐bound proteins: Experimental designs and applications

Article type: Research Article

Authors: Ruan, Ke‐He

Affiliations: Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, 6431 Fannin St., Houston, TX 77030, USA Tel.: +1 713 500 6769; Fax: +1 713 500 6810; E‐mail: kruan@uth.tmc.edu

Abstract: High resolution Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for determining the solution structures of peptides and small proteins, and their ligand binding functions. Molecular biology mutagenesis is a widely used and powerful approach for identification of the protein functions. We have developed a strategy integrating NMR experiments with mutagenesis studies to advance and extend the approaches used for structure/function relationship studies of proteins, especially for membrane‐bound proteins, which play important roles in physiopathological processes. The procedures include the design of the functional protein domain, identification of the solution structure and intermolecular contacts between the protein segment and its ligand. These determinations are resolved by high‐resolution 2D NMR spectroscopy, and followed by site‐directed mutagenesis of the residues suggested from the NMR experiment for the membrane‐bound proteins. The residues important to the protein functions, identified by the mutagenesis, were further used to re‐assign the NMR spectra and finalize the docking of the protein with its ligand. A structural model of the protein/ligand interaction can be constructed at an atomic level based on the NMR spectroscopy and mutagenesis results. As an application, the strategy has enhanced our knowledge in the understanding of the structure/function relationship for a membrane‐bound G protein coupling receptor, the thromboxane A2 receptor (TP receptor), interacting with its ligand, and a microsomal P450, prostacyclin synthase (PGIS), docking with its substrate in the endoplasmic reticulum (ER) membrane. In this review, we have summarized the principles and applications for this newly developed technique.

Journal: Spectroscopy, vol. 18, no. 1, pp. 13-29, 2004