This chapter discusses the use of isotopic labeling to assign the aromatic partners of cross-peaks of both wild-type and mutant proteins through the use of deuteration of both aliphatic and aromatic amino acid side chains. Comparison of the data for isotopically labeled wild-type and mutant proteins allow independent assignment of the cross-peaks in these proteins. This contribution will conclude with a description of how the side chain assignments can be extended to site-specific assignments. For a protein the size of Snase, 149 amino acids plus a hexapeptide tail in the form one study, methods that depend on resolved scalar couplings or on sufficient resolution of the two-dimensional data will typically not be entirely adequate. Although isotopic labeling is both expensive and labor intensive, it does yield unambiguous information about both wild-type and mutant proteins. In favorable cases assignments can be made by comparing the NMR data of wild-type and mutant proteins. However, comparative methods are limited to instances of conformationally silent amino acids, or when there is a localized probe of the protein structure.
ASJC Scopus subject areas
- Molecular Biology