[3] Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance

This chapter discusses the methods of biosynthetic enrichment of proteins with 15N in backbone and side-chain groups, as exemplified by the work with bacteriophage T4 lysozyme. Three necessary features for the investigation of 15N-labeled proteins are discussed. First, the protein must be rapidly and efficiently produced in milligram quantities. Second, the protein must be uniformly or selectively enriched in 15N by growth of the bacteria on defined media supplemented with the isotope. Third, new nuclear magnetic resonance (NMR) experiments have been developed, which either directly or indirectly yield information regarding the 15N nucleus or exploit the 15N as a means to filter 1H NMR spectra. One of the goals of the research on T4 lysozyme is to investigate the effect of amino acid substitutions on the structure and dynamics of the protein. Unfortunately, in three cases, it has been observed that many changes in the 15N- 1H spectra of uniformly 15N-labeled mutant lysozymes relative to the wild-type protein. This limits the extension of assignments of resonances from the wild-type to mutant lysozymes.