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 A2receptor (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.
Highlights
Biological functions of proteins are reflected in binding activities, such as an enzyme binding to its substrate and a receptor binding to its ligand
The detailed approaches used for our successful protein modeling of thromboxane A2 synthase (TXAS) [26,27], prostaglandin H2 synthase (PGHS) [28], prostacyclin synthase (PGIS) [29], and the TP receptor [30] can be used as references
circular dichroism (CD) and fluorescence experiments are especially useful because the approach is easy and the experimental conditions can be adopted for further Nuclear Magnetic Resonance (NMR) spectroscopic studies, and secondary structural observations in CD spectroscopy can be used as a structural reference for the NMR experiments
Summary
Biological functions of proteins are reflected in binding activities, such as an enzyme binding to its substrate and a receptor binding to its ligand. To identify the residues important to the ligand binding on the membrane proteins in structural terms is still a big challenge for the high resolution NMR spectroscopy. To take the advantages of the high resolution NMR spectroscopy, for the determination of the solution structure of the peptide, and site-directed mutagenesis, for the identification of the protein function at a single residue level, and combine the two approaches in a single system might enable the enlargement of the scope and enhancement of the powers of either approach individually. I introduce a way in my laboratory characterized by the structure/function relationship of membrane proteins using a system connecting the several approaches in one, which includes molecular modeling, constrained peptide synthesis, NMR structural determination, and site-directed mutagenesis.
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