2D AND 3D NMR-STUDY OF PHENYLALANINE RESIDUES IN PROTEINS BY REVERSE ISOTOPIC LABELING

A protein isotopic labeling strategy is presented which offers improved NMR sensitivity and resolution relative to the commonly used uniform C-13 labeling approach. Incorporation of specific residues at natural abundance into an otherwise fully C-13-enriched protein yields H-1 line widths for the unlabeled residues which are not adversely affected by C-13 and makes it possible to selectively focus on interactions between the unlabeled residues and the remainder of the protein. Modifications of C-13 editing and C-12 filtering procedures are described which optimize their sensitivity and resolution. The experiments are used to obtain complete assignments for all 10 phenylalanine aromatic spin systems in the DNA-binding domain of Drosophila heat shock factor and to obtain a large number of structurally important long-range NOE constraints. A novel J correlation experiment is also described which makes it possible to measure H-alpha-H-beta J couplings in larger proteins and yields quantitative values for all 10 phenylalanines in the DNA-binding domain of Drosophila heat shock factor.