16 April 1999

Ž .Chemical Physics Letters 303 1999 493–498

Determination of chemical shielding tensor of an indole carbonand 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, Australiab Department of Chemistry, Graduate School of Science, Kyoto UniÕersity, Kyoto 606-8502, Japan

c Physiology and Biophysics Department, Johns Hopkins UniÕersity, Baltimore, MD 21205, USAd 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 to4 3 3

the C´ –H dipolar axis was determined from the 13C chemical shiftr13C–1H dipolar 2D NMR powder pattern. The3Žprincipal values obtained were 208, 137 and 15 ppm with s perpendicular to the indole plane, and s least shielded33 11

.direction 58 off the C´ –H bond toward C . The side off the C´ –H bond was determined by comparing the reduced3 j3 3w 13 xchemical 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 tobiologically important molecules often rely on aknowledge of the chemical shift tensor: the principalvalues and the orientation of the chemical shift ten-sor for the site of interest provide information aboutmolecular structure and dynamics. The complete de-termination of the 13C shielding tensor for an aro-matic carbon in tryptophan is particularly importantfor interpreting 13C chemical shift anisotropies ob-

w xserved 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 powder13 Ž .sample C-enriched at the C C´ of the indole4 3

Ž .Scheme 1 , we have determined the principal valuesof the 13C chemical shift tensor and its orientationrelative to the C´ –H dipolar axis by a previously3

w xreported method 3 , which is the off-magic-angleŽ .spinning version of two-dimensional 2D separated

Ž . w xlocal field SLF spectroscopy 4 . The shieldingtensor orientation was confirmed by comparison witha molecular dynamics calculation of the reduced

Ž .chemical shift anisotropy CSA of a membranepeptide. Using the results, the tryptophan sidechain

Ž .orientations of gramicidin A gA aligned in phos-pholipid bilayer membranes were determined from

13 wthe C chemical shift anisotropies obtained for 4-13 xC-Trp gA. GA is a 15-residue peptide that acts asa monovalent cation channel and undergoes rotationaround 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

( )F. SeparoÕic et al.rChemical Physics Letters 303 1999 493–498494

Scheme 1.

w xclose to 1.0 5 . The tryptophans are important fordefining the conformation and orientation of the gAchannel within the lipid bilayer and for cation con-

w xductance 6 .

2. Experimental

L-tryptophan-indole-4-13C enriched to 99% waspurchased from Cambridge Isotope LaboratoriesŽ .Andover, MA, USA and used without further pu-rification. Aligned phospholipid bilayers incorporat-ing 13C labeled tryptophan gA were prepared as

w xdescribed 7 on ;35 glass coverslips within a 7mm glass tube and a phospholipid:gA molar ratio of15:1 and ;50 wt% water. Typically 7 mg of gAand 39 mg of dimyristoylphosphatidylcholineŽ . w 13DMPC were used. Three samples using 4- C-Trp-x w 13 x w 13 x9 gA, 4- C-Trp11 gA and 4- C-Trp13 gA were

prepared. Carbon-13 spectra were obtained undercross polarization conditions with high power decou-pling on a Bruker MSL-400 spectrometer operatingat 100.613 MHz for 13C. Spectra were acquired at

307 K with a pr2 pulse width of 7.5 ms, a mixingtime of 2 ms, a recycle time of 2 s and 35 000 scans.

The 13C chemical shiftr13C–1H dipolar 2D pow-der pattern was obtained by off-magic-angle spin-

w x Žning SLF spectroscopy 3 . The FSLG frequency-. w xshifted Lee–Goldburg -2 sequence 8 was used for

homonuclear dipolar decoupling. The off-magic an-gle was set to 61.68 throughout the experimental

w xsequence 9 . The experiments were performed on aChemagnetics CMX-300 NMR spectrometer operat-ing at 75.559 MHz for 13C with a 2 ms contact timeand a 3 s recycle time. The pr2 pulse for 1H was4.5 ms. The frequency switch for the FSLG-2 was"40 kHz and the FSLG pulse width was 14.7 ms.The sample spinning frequency was 4860 Hz.

A molecular dynamics trajectory of a gA:phos-Ž .pholipid system molar ratio 1:8 with 44 wt% water

was used to perform conformational samplingw x10,11 . The simulation system consists of 16 DMPC

Žlipid molecules surrounding a central gA channel a.gA dimer and solvated by 650 water molecules, a

total of 4385 atoms. An infinite membrane systemwas modeled by using an hexagonal boundary condi-tion within the bilayer plane, and a periodic bound-ary condition in the direction normal to the bilayer.The all-hydrogen potential function PARAM 22 ofthe CHARMM program was used for the gA andDMPC, and the TIP3P potential was used for thewater molecules.

3. Results and discussion

Experimental and simulated spectra of L-tryp-tophan-indole-4-13C are shown in Fig. 1. Fig. 1ashows the chemical shiftrdipolar 2D powder patternobtained while spinning at an off-magic angle u soff

1 2Ž61.68. Therefore, the CSA, is scaled by 3 cos uoff2

. 13 1y1 sy0.161, while the C– H dipolar couplingis further scaled by the scaling factor for the FSLG-2sequence. Although the latter is theoretically given tobe 1r63, for the frequency offset used, we deter-mined it to be 0.53 from the scaling of the Jcoupling in adamantane under MAS with FSLG-2decoupling for use in the analysis taking into accountexperimental nonidealities. Consequently, the dipolarcoupling is totally scaled by 0.085. The computersimulated spectrum is shown in Fig. 1b, and is in

( )F. SeparoÕic et al.rChemical Physics Letters 303 1999 493–498 495

Fig. 1. 13C chemical shiftr13C–1H dipolar 2D powder patterns ofŽ13 Ž . Ž .L-tryptophan C-enriched at the C C´ . a Experimental4 3

spectrum obtained under spinning at magic angle q6.98 and usingthe FSLG-2 pulse cycle for proton decoupling. The scaling factorsare 0.085 and y0.161 in the v and v dimensions, respectively.1 2

The spectral width is 4860 Hz in v and 152.8–85.2 ppm in v .1 2Ž .b Calculated spectrum taking into account the directly bondedand the next nearest-neighbour protons. The s orientation was33

assumed to be perpendicular to the indole plane and J equal toCH

160 Hz. The resulting shielding tensor values s , s and s11 22 33

were equal to 208, 137 15 ppm, respectively, with s 58 off the11Ž .C–H bond of the C C´ .4 3

good agreement with the experimental spectrum,Ž .where the most shielded direction s was as-33

sumed to be perpendicular to the indole plane. Fromthe simulation, the 13C principal chemical tensorvalues were determined to be s s208 ppm, s s11 22

137 ppm and s s15 ppm relative to TMS with33Ž .least shielded direction s 58 off the C–H bond.11

˚The C–H bond length was calculated to be 1.05 A in˚close comparison to 1.01 A obtained by X-ray crys-

w xtallography 12 . It is known that the C–H bondlength obtained by NMR exceeds that by X-ray tothis extent as a result of the influence of molecular

w xvibrations 13,14 .The gA molecule is rotating about its long axis,

making the CSA tensors axially symmetric aroundthe long axis. The chemical shift anisotropies, Dsss ys , of the tryptophan C carbons of Trp-9,5 H 4

Trp-11 and Trp-13 of gA in aligned bilayers weredetermined for the respective samples from the dif-ference in chemical shift position with the alignedsamples set at 08 and 908 to the magnetic field, i.e.the bilayer normal parallel and perpendicular to the

w 13field. As an example, spectra of a sample of 4- C-xTrp-13 gA in DMPC are shown in Fig. 2. The

results for Trp-9, Trp-11 and Trp-13 were y79,Ž .y70 and y77 "2 ppm, respectively. On the other

hand, we obtained the calculated CSA as follows: theinstantaneous gA conformations in aligned bilayers

w xwere obtained every 0.5 ps over 500 ps 10,11 bymolecular dynamics calculations. A snapshot of thegA conformation along the channel axis is shown inFig. 3. The instantaneous CSA of the tryptophan C4

carbons of gA were calculated using the moleculardynamics conformations and the above 4-13C tensorof L-tryptophan. The tensor orientation in the aminoacid was assumed to be the same in the peptide asthe sidechain is not directly involved in the forma-tion of the peptide bond. However, changes in H-bonding and intermolecular interactions that influ-ence tensor orientation will occur on going from adry amino acid powder to a hydrophobic membraneenvironment. The tensors were averaged with respectto the two gA monomers composing a dimer andwith time. However, we did not know which side 58

off the C´ –H bond was the least shielded direction,3

since SLF spectroscopy cannot determine the direc-tion either side of the bond. Therefore we calculatedthe average CSA for both directions. The calculated

( )F. SeparoÕic et al.rChemical Physics Letters 303 1999 493–498496

13 w 13 x Ž .Fig. 2. C static NMR spectra of 4- C-Trp-13 gA in aligned DMPC bilayers 1:15 at 307 K; Ø marks the resonance from the labeledŽ . Ž .tryptophan. The upper spectrum is with the bilayer normal parallel 08 , and the lower spectrum perpendicular 908 to the magnetic field.

( )F. SeparoÕic et al.rChemical Physics Letters 303 1999 493–498 497

Fig. 3. A snapshot of the gA ion channel axis taken during the 500 ps molecular dynamics simulation. To differentiate the two gAmonomers sitting one above the other, one molecule is darker.

results for Trp-9, Trp-11 and Trp-13 gA were y80,y65 and y93 ppm, respectively, for the s orien-11

tation on the C side, and y47, y31 and y51j3

ppm, respectively, for that on the other side. Obvi-ously, the former values are nearer to the experimen-tal results, fixing the s orientation to 58 off the11

C´ –H bond on the C side. Thus we were able to3 j3

obtain knowledge of the complete chemical shifttensor of the C´ carbon of tryptophan using SLF3

w 13 xspectroscopy for 4- C tryptophan together with13 w 13 xC static NMR of 4- C-Trp gA in aligned bilayersand molecular dynamics calculations.

The tryptophan sidechain orientations of gA inaligned bilayers from the molecular dynamics calcu-lations, and consistent with the experimental reducedCSA of the C´ carbon of gA tryptophans, was3

Ždetermined. The obtained tensor orientation u , u1 2.and u of the tryptophan C´ carbons of gA in3 3

aligned bilayers with respect to the channel axis isu s488 "98, u s428 "98 and u s808 "158,1 2 3

for the three tryptophans. This result indicates thatthe most shielded direction, s , is approximately33

perpendicular to the long axis of the gA channel. Theplane of the indole is almost parallel to the channelwith the indole N–H pointing toward the membranesurface. The orientations of the tryptophans relativeto the gA channel axis are close to those obtained by

w xseveral groups using NMR spectroscopy 15–17 .In the present study, we determined the complete

13C chemical shift tensor for the C´ of tryptophan,3

and applied it to obtain information on the trypto-phan orientations of gA in aligned phospholipidmembranes to demonstrate its usefulness. Knowl-edge of the full tensor of the tryptophan C´ carbon3

will be useful for protein structure determinationw x18 .

( )F. SeparoÕic et al.rChemical Physics Letters 303 1999 493–498498

References

w x Ž .1 S.J. Opella, Nat. Struct. Biol. 4S 1997 845.w x2 M. Schiffer, C.-H. Chang, F.J. Stevens, Protein Eng. 5

Ž .1992 213.w x Ž .3 T. Nakai, J. Ashida, T. Terao, J. Chem. Phys. 88 1988

6049.w x Ž .4 J.S. Waugh, Proc. Natl. Acad. Sci. USA 73 1976 1394.w x Ž .5 F. Separovic, R. Pax, B. Cornell, Mol. Phys. 78 1993 357.w x Ž .6 M. Cotton, F. Xu, T.A. Cross, Biophys. J. 73 1997 614.w x7 B.A. Cornell, F. Separovic, A.J. Baldassi, R. Smith, Biophys.

Ž .J. 53 1988 67.w x8 A. Bielecki, A.C. Kolbert, M.H. Levitt, Chem. Phys. Lett.

Ž .155 1989 341.w x Ž .9 T. Terao, H. Miura, A. Saika, J. Chem. Phys. 85 1986

3816.

w x Ž .10 T.B. Woolf, B. Roux, Proc. Natl. Acad. Sci. USA 91 199411631.

w x Ž .11 T.B. Woolf, B. Roux, Proteins 24 1996 92.w x Ž .12 R.A. Pasternak, Acta Crystallogr. 9 1956 341.w x Ž .13 E.R. Henry, A. Szabo, J. Chem. Phys. 82 1985 4753.w x Ž .14 Y. Ishii, T. Terao, J. Chem. Phys. 107 1997 2760.w x15 F. Separovic, K. Hayamizu, R. Smith, B.A. Cornell, Chem.

Ž .Phys. Lett. 181 1991 157.w x16 A.S. Arseniev, I.L. Barsukov, V.F. Bystrov, in: W. Voelter,

Ž .E. Bayer, Y.A. Ovchinikov, V.T. Ivanov Eds. , Chemistryof Peptides and Proteins, vol. 3, De Gruyter, Berlin, 1986.

w x Ž .17 W. Hu, N.D. Lazo, T.A. Cross, Biochemistry 34 199514138.

w x18 M. Ottiger, N. Tjandra, A. Bax, J. Am. Chem. Soc. 119Ž .1997 9825.