Sample optimization and identification of signal patterns of amino acid side chains in 2D RFDR spectra of the α-spectrin SH3 domain

Future structural investigations of proteins by solid-state CPP-MAS NMR will rely on uniformly labeled protein samples showing spectra with an excellent resolution. NMR samples of the solid alpha-spectrin SH3 domain were generated in four different ways, and their C-13 CPMAS spectra were compared. The spectrum of a [u-C-13, N-15]-labeled sample generated by precipitation shows very narrow C-13 signals and resolved scalar carbon-carbon couplings, Linewidths of 16-19 Hz were found for the three alanine CP signals of a selectively labeled [70% 3-C-13]alanine-enriched SH3 sample. The signal pattern of the isoleucine, of all prolines, valines, alanines, and serines, and of three of the four threonines were identified in 2D C-13-C-13 RFDR spectra of the [u-C-13, N-15]-labeled SH3 sample. A comparison of the C-13 chemical shifts of the found signal patterns with the C-13 assignment obtained in solution shows an intriguing match. (C) 2000 Academic Press.