Isotope Labeling of Proteins for NMR Spectroscopy Using Cell-Free Methods

Abstract

NMR spectroscopy is uniquely capable of providing information on the structure, function, and dynamics of proteins and other biomolecules in solution. Typically, milligram quantities of proteins are required, although recent technological improvements, including cryoprobes (see, for example, Serber et al. 2000) and high-field magnets (900 MHz), have improved the sensitivity of the method considerably. Recent experimental advances have also increased the molecular weight range assessable for study. With advanced techniques it is now possible to obtain limited information on complexes as large as 800 kDa (Riek et al. 2002). Isotopic labeling of the protein with 15N, 13C, 2H, or a combination of these isotopes is often required. In vitro protein synthesis is well suited to producing these types of samples. Herein we discuss the advantages of using in vitro methods to produce isotopically labeled proteins for NMR studies and show examples of the specialized information obtainable from such experiments.