Journal of Biomolecular NMR, 12: 259–276, 1998. KLUWER/ESCOM © 1998 Kluwer Academic Publishers. Printed in the Netherlands. 259 Some NMR experiments and a structure determination employing a { 15 N, 2 H} enriched protein T.K. Mal, S.J. Matthews a , H. Kovacs, I.D. Campbell & J. Boyd Oxford Centre for Molecular Sciences and Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K. a Department of Biochemistry, Imperial College of Science and Technology and Medicine, Exhibition Road, South Kensington, London, SW7 2AY, U.K. Received 26 November 1997; Accepted 26 March 1998 Key words: isotopic enrichment { 15 N, 2 H}, 1 H relaxation, structure calculations, isotope shifts, solvent interactions Abstract We present the results of studies of an aqueous sample of a highly { 15 N, 2 H} enriched protein, the SH3 domain from Fyn. Measurements of 1 H relaxation and interactions between H 2 O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances. The long-range isotope shifts are measured, for 1 H and 15 N, which arise due to perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this 1 H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7Å apart. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments. These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain. Abbreviations: 1D, one-dimensional; 2D, two-dimensional; 3D, three-dimensional; NOESY, nuclear Overhauser enhancement spectroscopy; HSQC, heteronuclear single quantum correlation; SA, simulated annealing; Tris- HCl, tris(hydroxymethyl)aminomethane-hydrochloride; SDS-PAGE, sodium dodecylsulphate-polyacrylamide gel eletrophoresis; PBS, buffered saline; CSA, chemical shift anisotropy; DSSP, define secondary structure of proteins. Introduction Several groups have explored the use of fully or par- tially 2 H isotopically enriched proteins (Crespi et al., 1968; Markley et al., 1968; Torchia et al., 1988; LeMaster et al., 1988; Tsang et al., 1990) in com- bination with 15 N enrichment (Grzesiek et al., 1995) and 15 N and 13 C enrichment (Grzesiek et al., 1993; Markus et al., 1994; Yamazaki et al., 1994; Farmer and Venters, 1995; Venters et al., 1995; Venters et al., 1996; Nietlispach et al., 1996; Gardner et al., 1997; Yamazaki et al., 1997; Sattler et al., 1997, Yu et al., 1997). The primary focus of these papers was to es- tablish a route to the structure determination of larger proteins than is possible with 15 N and 13 C isotopic enrichment alone. Examples are a Trp repressor-DNA complex (Zhang et al., 1994), Shc phosphotyrosine binding domain (Zhou et al., 1995), and the protein Bcl-X L (Muchmore et al., 1996). In this paper we use an aqueous sample of Fyn SH3 domain, where some effort was made to obtain as high a level of { 15 N, 2 H} enrichment as possible. The structure of the Fyn SH3 domain from the Fyn tyrosine kinase (Cooke and Perlmutter, 1989) has been deter- mined by both X-ray crystallography (Noble et al., 1993) and NMR (Morton et al., 1996) and shown to