5] Nuclear magnetic resonance spectroscopy of peptide ion channel ligands: Cloning and expression as aid to evaluation of structural and dynamic properties

Abstract

As candidates for structural measurements ion channels are daunting targets. Their large size and the fact that they are anchored in biological membranes make atomic resolution information unattainable by available techniques. There is a vast array of peptide ligands whose structures are more accessible that bind tightly to the outer vestibules of different ion channels. Peptides from this class are found in a variety of animal venoms and are characterized by high cysteine content and a conserved backbone fold. Structural information about these ligands is important for understanding their function as pharmacologic tools and as therapeutic entities. The high binding affinity and exquisite selectivity of these ligands suggests that structural inferences can be drawn about the ion channel from an adequate knowledge of the structure of the ligands themselves. Additionally, more direct information may be obtained about the ion channel by studying the ligand bound to a relevant fragment of the channel. These peptides are particularly well suited to nuclear magnetic resonance (NMR) measurements. This chapter briefly discusses the aspects of NMR spectroscopy pertinent to the study of peptide toxins that bind to ion channels, and detail an expression and purification protocol that will both enable NMR-active isotope incorporation into these peptides and provide a facile system for producing mutant analogs for structure-function studies. The chapter discusses experimental considerations for carrying out NMR studies on this class of peptides.