Biosynthetic Incorporation of15N and13C for Assignment and Interpretation of Nuclear Magnetic Resonance Spectra of Proteins

Abstract

The use of isotopic substitution is a time-honoured method for simplifying the nuclear magnetic resonance spectra of biological macromolecules. For example, the biosynthetic incorporation of a heteronucleus such as 15 N or 13 C into a specific amino acid residue in a protein followed by direct observation of the 15 N or 13 C NMR spectrum could provide a means to specifically observe a given amino acid type in that protein. By observation of the chemical shift or relaxation properties as a function of pH, ligand concentration, etc. a number of important conclusions concerning the pK a values of specific residues, the affinity of the protein for various ligands, or dynamic properties of the protein can be deduced. (See Henry et al.1986 a,b; 1987 for an elegant modern example). In such situations, direct observation of the heteronucleus is a powerful means to observe environmental changes (Niu et al.1979) but often these measurements are not readily interpretable in terms of alterations of protein structure. Although proton-proton dipolar interactions (NOEs) typically provide the richest source of such structural information, these interactions are not monitored in most experiments which directly observe the heteronucleus.