Solid-state NMR study of the SH3 domain of ?-spectrin: application of13C-15N TEDOR and REDOR

Abstract

A fully 13 C- 15 N-labeled and a selectively alanine- 13 C tryptophan- 15 N ring -labeled sample of the Src homology region 3 (SH3) domain of -spectrin (chicken), a 62 residue protein, were biosynthesized and studied by solid-state cross-polarization magic angle spinning (CP/MAS) NMR, 13 C- 15 N rotational echo double resonance (REDOR) and 15 N- 13 C transferred echo double resonance (TEDOR) spectroscopy. In the first part of the study it is shown that spectral editing with the TEDOR sequence leads to a drastic simplification of the 13 C MAS spectrum of the fully labeled sample, allowing the resolved spectroscopy of groups of 13 C nuclei, according to their distance to neighboring 15 N nuclei. In the second part of the study the inter-residual distance between the alanine residue Ala55 and the tryptophan residue Trp42 was determined by the measurement of the dipolar coupling between Ala- 13 C and Trp- 15 N ring , yielding a dipolar coupling of 48 8 Hz, which after correction for fast molecular vibrations gives a value of 53 8 Hz, corresponding to a CN distance of 3:85 0:25 Hz. The result is compared to the CN distances obtained by x-ray diffraction and liquid-state NMR. Copyright © 2000 John Wiley & Sons, Ltd.