16 April 1999 Ž . Chemical Physics Letters 303 1999 493–498 Determination of chemical shielding tensor of an indole carbon and application to tryptophan orientation of a membrane peptide Frances Separovic a, ) , Jun Ashida a,1 , Tom Woolf c , Ross Smith d , Takehiko Terao b a School of Chemistry, UniÕersity of Melbourne, ParkÕille, Vic. 3052, Australia b Department of Chemistry, Graduate School of Science, Kyoto UniÕersity, Kyoto 606-8502, Japan c Physiology and Biophysics Department, Johns Hopkins UniÕersity, Baltimore, MD 21205, USA d Biochemistry Department, UniÕersity of Queensland, Brisbane, Qld. 4072 Australia Received 13 October 1998; in final form 16 February 1999 Abstract 13 Ž . Using tryptophan C-enriched at the C C ´ of the indole, the orientation of the C ´ chemical shift tensor relative to 4 3 3 the C ´ –H dipolar axis was determined from the 13 C chemical shiftr 13 C– 1 H dipolar 2D NMR powder pattern. The 3 Ž principal values obtained were 208, 137 and 15 ppm with s perpendicular to the indole plane, and s least shielded 33 11 . direction 58 off the C ´ –H bond toward C . The side off the C ´ –H bond was determined by comparing the reduced 3 j 3 3 w 13 x chemical shift anisotropies obtained by solid-state NMR and from molecular dynamics calculations of 4- C tryptophans in gramicidin A aligned in phospholipid membranes. q 1999 Elsevier Science B.V. All rights reserved. 1. Introduction Applications of solid-state NMR spectroscopy to biologically important molecules often rely on a knowledge of the chemical shift tensor: the principal values and the orientation of the chemical shift ten- sor for the site of interest provide information about molecular structure and dynamics. The complete de- termination of the 13 C shielding tensor for an aro- matic carbon in tryptophan is particularly important for interpreting 13 C chemical shift anisotropies ob- w x served for membrane proteins and peptides 1,2 . ) Corresponding author. Fax: q61 3 9347 5180; e-mail: f.separovic@chemistry.unimelb.edu.au 1 Present address: Varian Japan, Sumitomo Shibaura Bldg., 4-16-36 Shibaura, Minato-ku, Tokyo 108-0023, Japan. In the present work, using a tryptophan powder 13 Ž . sample C-enriched at the C C ´ of the indole 4 3 Ž . Scheme 1 , we have determined the principal values of the 13 C chemical shift tensor and its orientation relative to the C ´ –H dipolar axis by a previously 3 wx reported method 3 , which is the off-magic-angle Ž . spinning version of two-dimensional 2D separated Ž . wx local field SLF spectroscopy 4 . The shielding tensor orientation was confirmed by comparison with a molecular dynamics calculation of the reduced Ž . chemical shift anisotropy CSA of a membrane peptide. Using the results, the tryptophan sidechain Ž . orientations of gramicidin A gA aligned in phos- pholipid bilayer membranes were determined from 13 w the C chemical shift anisotropies obtained for 4- 13 x C-Trp gA. GA is a 15-residue peptide that acts as a monovalent cation channel and undergoes rotation around the channel axis with an order parameter 0009-2614r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0009-2614 99 00253-5