- Email: [email protected]
Nitrogen-15 chemical shift anisotropy and 1H–15N dipolar coupling tensors associated with the phenylalanine residue in the solid state
13 August 1999
Chemical Physics Letters 309 Ž1999. 209–214 www.elsevier.nlrlocatercplett
Nitrogen-15 chemical shift anisotropy and 1 H– 15 N dipolar coupling tensors associated with the phenylalanine residue in the solid state D.K. Lee a
a,b
, J.S. Santos a , A. Ramamoorthy
a,b,)
Biophysics Research DiÕision, The UniÕersity of Michigan, Ann Arbor, MI 48109-1055, USA b Department of Chemistry, The UniÕersity of Michigan, Ann Arbor, MI 48109-1055, USA Received 12 May 1999; in final form 3 June 1999
Abstract Nitrogen-15 chemical shift anisotropy ŽCSA. and 1 H– 15 N dipolar coupling tensors associated with the Phe-16 residue of the magainin2 peptide are reported in this Letter. The experimental results predict that the magnitudes of the 15 N CSA tensor are s 11N s 55 " 2, s 22N s 80 " 2 and s 33N s 220 " 2 ppm. The results also suggest that the least shielded element, s 33N , is in the peptide plane making an angle of 22 " 38 with the N–H bond vector whereas s 11N and s 22N are 45 " 158 away from the peptide plane and the normal to the peptide plane, respectively. The magnitudes of the principal elements of the 15 N CSA tensors associated with 15 N-Phe-16 and 15 N-Gly-18 sites of the magainin2 peptide are significantly different while the orientation of the tensors in the molecular frame is the same. q 1999 Elsevier Science B.V. All rights reserved.
1. Introduction Recently there has been considerable interest in spin interaction tensors pertaining to the amide fragments of peptides and proteins that are determined by using solid-state NMR techniques w1–10x. The magnitudes and orientations of the principal components of the 15 N chemical shift anisotropy ŽCSA. tensor are intimately related to the local nuclear environment. For example, variations in the primary and secondary structures, and intermolecular hydrogen-bonding are among the properties that are thought to be responsible for observed variations in 15 N CSA tensors. This has resulted in studies focusing on the ) Corresponding author. Fax: q1 734 764 8776; e-mail: [email protected]
relationship between the chemical shift and the structural characteristics of polypeptides w2,11x. Knowledge of the 15 N CSA tensors is also important in studies of cross correlation, polypeptide dynamics, and to determine the structure of oriented and disordered systems. Recent developments in experimental NMR spectroscopy w12,13x and computational chemistry w14x have led to an enhanced interest in the application of CSA tensors. The purpose of this Letter is twofold: Ž1. to determine the 15 N CSA tensor of a phenylalanine residue, and Ž2. to further examine the variation of the 15 N CSA tensor in various amino acid residues of polypeptides that have the same backbone conformation. We have chosen a synthetic magainin2 peptide in the solid state enriched with 15 N isotope at the phenylalanine amide site for this study as the conformation of this
0009-2614r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 Ž 9 9 . 0 0 6 8 9 - 2
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peptide is reported in the literature w15,16x. Here, we report both magnitude and orientation of the principal components of the 15 N CSA and 1 H– 15 N dipolar coupling tensors. A one-dimensional dipolar-shift solid-state NMR technique was used in this study. The details of the technique and the procedure to characterize the tensors are given elsewhere w13x.
2. Experimental Fmoc-phenylalanine amino acid labeled with 15 N was purchased from Cambridge Isotope Laboratory ŽCambridge, MA.. All other amino acids were purchased from PerSeptive Biosystems ŽFramingham, MA . and used without further purification. The magainin2 peptide Ž n-GIGKFLHSAKKFGKAFVGEIMNS-amide. was synthesized with 15 N-phenylalanine at position 16 using fluorenylmethoxycarbonyl ŽFmoc. blocking chemistry and an automated solid-phase peptide synthesizer. All of the experiments were performed on a Chemagnetics Infinity 400 MHz solid-state NMR spectrometer operating at a field of 9.4 T with resonance frequencies 400.14 and 40.54 MHz for 1 H and 15 N, respectively. All of the magic angle spinning ŽMAS. experiments were performed using a Chemagnetics double resonance MAS probe. The powder peptide sample was ground and packed into zirconia rotors Ž5 mm o.d... Typical 908 pulse lengths were 3.0–3.7 and 3.5–4.8 ms for 1 H and 15 N, respectively. Cross-polarization ŽCP. was achieved under the Hartmann–Hahn matching condition with an optimum contact time of 2 ms. Following CP, the 15 N magnetization was refocused by a 1808 pulse to overcome the difficulties due to the receiver dead time and the second-half of the spin-echo signal was acquired. For experiments under MAS conditions, a two-pulse phase modulated ŽTPPM. decoupling sequence w17x was used to decouple protons during the 15 N signal acquisition. A recycle delay of 5 s was used. For the one-dimensional dipolar-chemical shift experiment w13x, the frequency of proton decoupling was shifted by an offset 54.8 kHz in order to establish an effective field at the magic angle w13x. This magic angle rf irradiation of protons suppresses the 1 H– 1 H homonuclear dipolar interactions and thus the resultant one-dimensional spectrum consists of the 15 N
CSA and 1 H– 15 N dipolar interactions. An experimentally determined scaling factor, 0.56 " 0.02, for the magic angle rf irradiation was used in the calculations of the dipolar-shift spectra. All the 15 N spectra were referenced with respect to external saturated aqueous NH 4 Cl solution, which has a chemical shift of 27.3 ppm relative to NH 3 Žliquid, 258C.. Calculations of the one-dimensional chemical shift and dipolar-shift powder patterns were carried out as explained in our previous publication w13x.
3. Results and discussion The static experimental 15 N chemical shift spectrum of magainin2 peptide labeled with 15 N-Phe amino acid is shown in Fig. 1A Žsolid line.. The powder pattern is found to be a best-fit with a calculated spectrum Ždashed lines in Fig. 1A. obtained using the following values for the principal elements of the 15 N chemical shift interaction: s 11N s 55 " 2, s 22N s 80 " 2 and s 33N s 220 " 2 ppm. The MAS spectrum of the same sample is shown in Fig. 1C. A 6 ppm line width of the isotropic 15 N chemical shift line at 119 ppm in Fig. 1C may be attributed to the presence of multiple crystal forms of the peptide. This may be the reason for the broad
Fig. 1. Nitrogen-15 chemical shift ŽA. and 1 H– 15 N dipolar-15 Nshift ŽB. spectra of a powder sample of w15 N-Phe-16x-magainin2 peptide. Experimental and simulated spectra are given in solid and dashed lines, respectively. ŽC. The magic angle spinning spectrum of the peptide.
D.K. Lee et al.r Chemical Physics Letters 309 (1999) 209–214
shoulders in the powder pattern in Fig. 1A and hence the uncertainty in the measurement of the magnitudes of the principal components of the 15 N chemical shift tensor. The experimental spectrum consisting of the 15 N chemical shift as well as the 1 H– 15 N dipolar coupling interactions Žcalled as dipolar-shift spectrum. is shown in Fig. 1B Žsolid line.. This spectrum was obtained as explained in the experimental section of this Letter. The best-fitting calculated spectrum presented in dashed lines in Fig. 1B was obtained using the following parameters: s 11N s 55 " 2 ppm, s 22N s 80 " 2 ppm, s 33N s 220 " 2 ppm, a N s 45 " 158, b N s 22 " 38 and a N–H bond ˚ In order to determine length of 1.06 " 0.01 A. the presence of any molecular motions in the peptide that average the chemical shift and dipolar coupling interactions, experiments were also performed at y508C. There was no difference in the parameters determined from the low-temperature and room-temperature spectra. Thus, the reported results in this study are rigid 15 N CSA and 1 H– 15 N dipolar coupling tensors of the 15 N-Phe residue in the magainin2 peptide. The dependence of the frequencies at which the shoulders occur in the simulated dipolar-shift spectrum Žsee Fig. 1B. on the values of the angles a N and b N is shown in Fig. 2. For comparison, the experimentally determined frequency values are shown in solid dots with an error bar. The dipolarshift spectrum is highly sensitive to the angle b N Žsee Fig. 2A. and therefore the angle was determined with relatively high accuracy. On the other hand, since the value of the angle b N is small, the dipolar-shift spectrum is relatively less sensitive to a N values Žsee Fig. 2B. and therefore a large error is reported in the measurement of the a N angle. Based on a single crystal study reported in the literature w18x, the least shielding tensor element, s 33N , is expected to be in the peptide plane of the Phe-16 residue of magainin2 and is 228 away from the direction of the N–H bond. Using the quantum calculation results w14x, it is predicted that s 33N is tilted by 228 towards the N–CO bond in the peptide plane. Based on the symmetry arguments, the most shielding element of the tensor, s 11N , is expected to be oriented on or near the peptide plane while s 22N is expected to be oriented along or near the normal to the peptide plane. The angle a N determined from
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this study suggests that s 11N is 458 away from the peptide plane while s 22N is 458 away from the normal to the peptide plane. This prediction, within the experimental error, is in good agreement with recent reports in the literature w10,12,13x. On the other hand, it differs from those studies that reported that s 11N is in the peptide plane and s 22N is along the normal to the peptide plane w19–24x. According to a study on the quantum mechanical calculations of 15 N shielding tensors in peptides w14x, the extent of tilt of s 11N from the peptide plane and s 22N from the normal to the peptide plane depends on the dihedral angle, c , of the molecular backbone of the peptide w14x. Furthermore, for c s y608 to 608, s 11N and s 22N rotate about the s 33N axis with a maximum deviation of 458 for the s 11N axis from the peptide plane or s 22N axis from the normal to the peptide plane. The experimental results reported in this Letter are consistent with the theoretical calculations reported in the literature w14x. The N–H bond ˚ determined from this length r NH s 1.06 " 0.01 A study is also in agreement with the previous NMR studies w10x. However, this value is longer than that determined using diffraction studies w25x. This difference may be attributed to molecular vibrations present in the crystal lattice that partially averages the 1 H– 15 N dipolar coupling measured using solid-state NMR methods w26x. It may be mentioned here that the isotropic 15 N chemical shift frequency of the Phe residue in polypeptides in solution ranges from 110.5 to 131.5 ppm with an average of 120.7 ppm. Also, solid-state NMR studies on n-acetylw1-13 CxGly-Lw15 Nx-PheNH 2 powder sample predicted three different 15 N isotropic chemical values Ž119, 121.4 and 123.3 ppm. from three magnetically inequivalent lattice environments w23,24x. These reported values are in good agreement with the value, 119 ppm, measured from the CPMAS spectrum in Fig. 1C. The possible sources of experimental errors of tensor values reported in this Letter could be the presence of multiple peptide conformations, molecular vibrations, and a variation Ž"0.02. in the scaling factor of the Lee–Goldburg decoupling sequence. It is worth pointing out that the magnitudes of the 15 N chemical shift tensors of 15 N-Phe-16 and 15 NGly-18 residues in the same magainin2 peptide are significantly different while the orientation of the tensors is the same within the experimental errors.
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Fig. 2. Variation of the frequency of the shoulders in the simulated dipolar-shift spectrum of a powder sample of w15 N-Phe-16x-magainin2 peptide with the values of: ŽA. b N and ŽB. a N angles. Experimentally determined frequency values are shown in solid dots with an error bar.
The tensor parameters determined for the 15 N-Gly-18 residue are: s 11N s 42 " 2 ppm, s 22N s 73 " 2 ppm,
s 33N s 215 " 2 ppm, a N s 30 " 108 and b N s 22 " 28. Since the backbone conformation of the maga-
D.K. Lee et al.r Chemical Physics Letters 309 (1999) 209–214
inin2 peptide can be assumed to be the same in the sites of these two residues, the difference in the tensors could be attributed to the difference in the electronic environments at the 15 N nuclei in these residues. The similarity of a N values for the 15 N chemical shift tensors at Phe-16 and Gly-18 sites of the magainin2 peptide further confirms the theoretical prediction that the angle a N mostly depends on the dihedral angle c . The 15 N CSA tensors reported in this Letter are also significantly different from those determined from the amide sites of model peptides or small peptides in solids w10x. Therefore, care must be taken in the determination of the molecular backbone conformation of uniaxially oriented polypeptides, characterization of molecular backbone dynamics, and analysis of relaxation data using CSA tensors obtained from model peptides. Since it is not possible to characterize the spin interaction tensors of all amide sites in a protein, it would be fruitful to generate a large collection of experimental data in order to show the variation of CSA tensors on the primary and the secondary structures of polypeptides for the structural studies on proteins.
4. Conclusions Rigid 15 N chemical shift and 1 H– 15 N dipolar coupling tensors of 15 N-Phe residue in a powder sample of the magainin2 peptide have been successfully characterized using a simple one-dimensional dipolar-shift solid-state NMR method. The 15 N CSA tensor is significantly different from that of other amino acid residues in polypeptides. Based on the theoretical calculations and as suggested by the experimental results in this Letter, the deviation of the s 11N axis from the peptide plane and the s 22N axis from the normal to the peptide plane depends on the dihedral angle c of the peptide. These results should be useful in the structure determination of uniaxially oriented membrane-bound polypeptides or proteins using solid-state NMR spectroscopy w27,28x, to study partially aligned bicelles w29x using high-resolution solution NMR methods, and to examine the cross correlation studies in globular proteins w2–9,30x.
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Acknowledgements Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. This research was supported in part by the research funds from the Horace H. Rackham School of Graduate Studies and Michigan memorial Phoenix project at the University of Michigan. Funding support from the Cambridge Isotope Laboratories is also acknowledged.
References w1x W.P. Power, R.E. Wasylishen, Annu. Rep. NMR Spectrosc. 23 Ž1991. 1. w2x A. Shoji, S. Ando, S. Kuroki, I. Ando, G.A. Webb, Annu. Rep. NMR Spectrosc. 26 Ž1993. 55. w3x N. Tjandra, A. Szabo, A. Bax, J. Am. Chem. Soc. 118 Ž1996. 6986. w4x N. Tjandra, A. Bax, J. Am. Chem. Soc. 119 Ž1997. 9576. w5x K. Pervushin, R. Riek, G. Wider, K. Wuthrich, Proc. Natl. Acad. Sci. USA 94 Ž1997. 12366. w6x M.W.F. Fischer, L. Zeng, Y.X. Pang, W.D. Hu, A. Majumdar, E.R.P. Zuiderweg, J. Am. Chem. Soc. 119 Ž1997. 12629. w7x J. Boyd, C. Redfield, J. Am. Chem. Soc. 120 Ž1998. 9692. w8x D. Fushman, N. Tjandra, D. Cowburn, J. Am. Chem. Soc. 120 Ž1998. 10947. w9x D. Fushman, D. Cowburn, J. Biomol. NMR 13 Ž1999. 139. w10x D.K. Lee, R.J. Wittebort, A. Ramamoorthy, J. Am. Chem. Soc. 120 Ž1998. 8868, and references cited therein. w11x D.K. Lee, A. Ramamoorthy, J. Phys. Chem. B 103 Ž1999. 271. w12x C.H. Wu, A. Ramamoorthy, L.M. Gierasch, S.J. Opella, J. Am. Chem. Soc. 117 Ž1995. 6148. w13x D.K. Lee, A. Ramamoorthy, J. Magn. Reson. 133 Ž1998. 204. w14x A.E. Walling, R.E. Pargas, A.C. deDios, J. Phys. Chem. A 101 Ž1997. 7299. w15x A. Ramamoorthy, F.M. Marassi, M. Zasloff, S.J. Opella, J. Biomol. NMR 6 Ž1995. 329. w16x J. Gesell, M. Zasloff, S.J. Opella, J. Biomol. NMR 9 Ž1997. 127. w17x A.E. Bennett, C.M. Rienstra, M. Auger, K.V. Lakshmi, R.G. Griffin, J. Chem. Phys. 103 Ž1995. 6951. w18x G.S. Harbison, L.W. Jelinski, R.E. Stark, D.A. Torchia, J. Herzfeld, R.G. Griffin, J. Magn. Reson. 60 Ž1984. 79. w19x M. Munowitz, R.G. Griffin, G. Bodenhausen, T.H. Huang, J. Am. Chem. Soc. 103 Ž1980. 2529. w20x J.E. Roberts, G.S. Harbison, M.G. Munowitz, J. Herzfeld, R.G. Griffin, J. Am. Chem. Soc. 109 Ž1987. 4163.
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w21x Y. Hiyama, C.H. Niu, J.V. Silverton, A. Bavoso, D.A. Torchia, J. Am. Chem. Soc. 110 Ž1988. 2378. w22x W. Mai, W. Hu, T.A. Cross, Protein Sci. 2 Ž1993. 532. w23x T.G. Oas, C.J. Hartzell, F.W. Dahlquist, G. Drobny, J. Am. Chem. Soc. 109 Ž1987. 5962. w24x C.J. Hartzell, M. Whitfield, T.G. Oas, G.P. Drobny, J. Am. Chem. Soc. 109 Ž1987. 5966. w25x A. Kvick, A.R. Al-Karaghouli, T.F. Koetzle, Acta Crystallogr. B 33 Ž1977. 3796.
w26x T. Nakai, J. Ashida, T. Terao, Mol. Phys. 67 Ž1989. 839. w27x T.A. Cross, S.J. Opella, Curr. Opin. Struct. Biol. 4 Ž1994. 574. w28x D.E. Warschawski, M. Traikia, P.F. Devaux, G. Bodenhausen, Biochimie 80 Ž1998. 437. w29x C.R. Sanders, B.J. Hare, K.P. Howard, J. Prestegard, Prog. NMR Spectrosc. 26 Ž1993. 421. w30x D.W. Yang, A. Mittermaier, Y.K. Mok, L.E. Kay, J. Mol. Biol. 276 Ž1998. 939.
Chemical Physics Letters 309 Ž1999. 209–214 www.elsevier.nlrlocatercplett
Nitrogen-15 chemical shift anisotropy and 1 H– 15 N dipolar coupling tensors associated with the phenylalanine residue in the solid state D.K. Lee a
a,b
, J.S. Santos a , A. Ramamoorthy
a,b,)
Biophysics Research DiÕision, The UniÕersity of Michigan, Ann Arbor, MI 48109-1055, USA b Department of Chemistry, The UniÕersity of Michigan, Ann Arbor, MI 48109-1055, USA Received 12 May 1999; in final form 3 June 1999
Abstract Nitrogen-15 chemical shift anisotropy ŽCSA. and 1 H– 15 N dipolar coupling tensors associated with the Phe-16 residue of the magainin2 peptide are reported in this Letter. The experimental results predict that the magnitudes of the 15 N CSA tensor are s 11N s 55 " 2, s 22N s 80 " 2 and s 33N s 220 " 2 ppm. The results also suggest that the least shielded element, s 33N , is in the peptide plane making an angle of 22 " 38 with the N–H bond vector whereas s 11N and s 22N are 45 " 158 away from the peptide plane and the normal to the peptide plane, respectively. The magnitudes of the principal elements of the 15 N CSA tensors associated with 15 N-Phe-16 and 15 N-Gly-18 sites of the magainin2 peptide are significantly different while the orientation of the tensors in the molecular frame is the same. q 1999 Elsevier Science B.V. All rights reserved.
1. Introduction Recently there has been considerable interest in spin interaction tensors pertaining to the amide fragments of peptides and proteins that are determined by using solid-state NMR techniques w1–10x. The magnitudes and orientations of the principal components of the 15 N chemical shift anisotropy ŽCSA. tensor are intimately related to the local nuclear environment. For example, variations in the primary and secondary structures, and intermolecular hydrogen-bonding are among the properties that are thought to be responsible for observed variations in 15 N CSA tensors. This has resulted in studies focusing on the ) Corresponding author. Fax: q1 734 764 8776; e-mail: [email protected]
relationship between the chemical shift and the structural characteristics of polypeptides w2,11x. Knowledge of the 15 N CSA tensors is also important in studies of cross correlation, polypeptide dynamics, and to determine the structure of oriented and disordered systems. Recent developments in experimental NMR spectroscopy w12,13x and computational chemistry w14x have led to an enhanced interest in the application of CSA tensors. The purpose of this Letter is twofold: Ž1. to determine the 15 N CSA tensor of a phenylalanine residue, and Ž2. to further examine the variation of the 15 N CSA tensor in various amino acid residues of polypeptides that have the same backbone conformation. We have chosen a synthetic magainin2 peptide in the solid state enriched with 15 N isotope at the phenylalanine amide site for this study as the conformation of this
0009-2614r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 Ž 9 9 . 0 0 6 8 9 - 2
210
D.K. Lee et al.r Chemical Physics Letters 309 (1999) 209–214
peptide is reported in the literature w15,16x. Here, we report both magnitude and orientation of the principal components of the 15 N CSA and 1 H– 15 N dipolar coupling tensors. A one-dimensional dipolar-shift solid-state NMR technique was used in this study. The details of the technique and the procedure to characterize the tensors are given elsewhere w13x.
2. Experimental Fmoc-phenylalanine amino acid labeled with 15 N was purchased from Cambridge Isotope Laboratory ŽCambridge, MA.. All other amino acids were purchased from PerSeptive Biosystems ŽFramingham, MA . and used without further purification. The magainin2 peptide Ž n-GIGKFLHSAKKFGKAFVGEIMNS-amide. was synthesized with 15 N-phenylalanine at position 16 using fluorenylmethoxycarbonyl ŽFmoc. blocking chemistry and an automated solid-phase peptide synthesizer. All of the experiments were performed on a Chemagnetics Infinity 400 MHz solid-state NMR spectrometer operating at a field of 9.4 T with resonance frequencies 400.14 and 40.54 MHz for 1 H and 15 N, respectively. All of the magic angle spinning ŽMAS. experiments were performed using a Chemagnetics double resonance MAS probe. The powder peptide sample was ground and packed into zirconia rotors Ž5 mm o.d... Typical 908 pulse lengths were 3.0–3.7 and 3.5–4.8 ms for 1 H and 15 N, respectively. Cross-polarization ŽCP. was achieved under the Hartmann–Hahn matching condition with an optimum contact time of 2 ms. Following CP, the 15 N magnetization was refocused by a 1808 pulse to overcome the difficulties due to the receiver dead time and the second-half of the spin-echo signal was acquired. For experiments under MAS conditions, a two-pulse phase modulated ŽTPPM. decoupling sequence w17x was used to decouple protons during the 15 N signal acquisition. A recycle delay of 5 s was used. For the one-dimensional dipolar-chemical shift experiment w13x, the frequency of proton decoupling was shifted by an offset 54.8 kHz in order to establish an effective field at the magic angle w13x. This magic angle rf irradiation of protons suppresses the 1 H– 1 H homonuclear dipolar interactions and thus the resultant one-dimensional spectrum consists of the 15 N
CSA and 1 H– 15 N dipolar interactions. An experimentally determined scaling factor, 0.56 " 0.02, for the magic angle rf irradiation was used in the calculations of the dipolar-shift spectra. All the 15 N spectra were referenced with respect to external saturated aqueous NH 4 Cl solution, which has a chemical shift of 27.3 ppm relative to NH 3 Žliquid, 258C.. Calculations of the one-dimensional chemical shift and dipolar-shift powder patterns were carried out as explained in our previous publication w13x.
3. Results and discussion The static experimental 15 N chemical shift spectrum of magainin2 peptide labeled with 15 N-Phe amino acid is shown in Fig. 1A Žsolid line.. The powder pattern is found to be a best-fit with a calculated spectrum Ždashed lines in Fig. 1A. obtained using the following values for the principal elements of the 15 N chemical shift interaction: s 11N s 55 " 2, s 22N s 80 " 2 and s 33N s 220 " 2 ppm. The MAS spectrum of the same sample is shown in Fig. 1C. A 6 ppm line width of the isotropic 15 N chemical shift line at 119 ppm in Fig. 1C may be attributed to the presence of multiple crystal forms of the peptide. This may be the reason for the broad
Fig. 1. Nitrogen-15 chemical shift ŽA. and 1 H– 15 N dipolar-15 Nshift ŽB. spectra of a powder sample of w15 N-Phe-16x-magainin2 peptide. Experimental and simulated spectra are given in solid and dashed lines, respectively. ŽC. The magic angle spinning spectrum of the peptide.
D.K. Lee et al.r Chemical Physics Letters 309 (1999) 209–214
shoulders in the powder pattern in Fig. 1A and hence the uncertainty in the measurement of the magnitudes of the principal components of the 15 N chemical shift tensor. The experimental spectrum consisting of the 15 N chemical shift as well as the 1 H– 15 N dipolar coupling interactions Žcalled as dipolar-shift spectrum. is shown in Fig. 1B Žsolid line.. This spectrum was obtained as explained in the experimental section of this Letter. The best-fitting calculated spectrum presented in dashed lines in Fig. 1B was obtained using the following parameters: s 11N s 55 " 2 ppm, s 22N s 80 " 2 ppm, s 33N s 220 " 2 ppm, a N s 45 " 158, b N s 22 " 38 and a N–H bond ˚ In order to determine length of 1.06 " 0.01 A. the presence of any molecular motions in the peptide that average the chemical shift and dipolar coupling interactions, experiments were also performed at y508C. There was no difference in the parameters determined from the low-temperature and room-temperature spectra. Thus, the reported results in this study are rigid 15 N CSA and 1 H– 15 N dipolar coupling tensors of the 15 N-Phe residue in the magainin2 peptide. The dependence of the frequencies at which the shoulders occur in the simulated dipolar-shift spectrum Žsee Fig. 1B. on the values of the angles a N and b N is shown in Fig. 2. For comparison, the experimentally determined frequency values are shown in solid dots with an error bar. The dipolarshift spectrum is highly sensitive to the angle b N Žsee Fig. 2A. and therefore the angle was determined with relatively high accuracy. On the other hand, since the value of the angle b N is small, the dipolar-shift spectrum is relatively less sensitive to a N values Žsee Fig. 2B. and therefore a large error is reported in the measurement of the a N angle. Based on a single crystal study reported in the literature w18x, the least shielding tensor element, s 33N , is expected to be in the peptide plane of the Phe-16 residue of magainin2 and is 228 away from the direction of the N–H bond. Using the quantum calculation results w14x, it is predicted that s 33N is tilted by 228 towards the N–CO bond in the peptide plane. Based on the symmetry arguments, the most shielding element of the tensor, s 11N , is expected to be oriented on or near the peptide plane while s 22N is expected to be oriented along or near the normal to the peptide plane. The angle a N determined from
211
this study suggests that s 11N is 458 away from the peptide plane while s 22N is 458 away from the normal to the peptide plane. This prediction, within the experimental error, is in good agreement with recent reports in the literature w10,12,13x. On the other hand, it differs from those studies that reported that s 11N is in the peptide plane and s 22N is along the normal to the peptide plane w19–24x. According to a study on the quantum mechanical calculations of 15 N shielding tensors in peptides w14x, the extent of tilt of s 11N from the peptide plane and s 22N from the normal to the peptide plane depends on the dihedral angle, c , of the molecular backbone of the peptide w14x. Furthermore, for c s y608 to 608, s 11N and s 22N rotate about the s 33N axis with a maximum deviation of 458 for the s 11N axis from the peptide plane or s 22N axis from the normal to the peptide plane. The experimental results reported in this Letter are consistent with the theoretical calculations reported in the literature w14x. The N–H bond ˚ determined from this length r NH s 1.06 " 0.01 A study is also in agreement with the previous NMR studies w10x. However, this value is longer than that determined using diffraction studies w25x. This difference may be attributed to molecular vibrations present in the crystal lattice that partially averages the 1 H– 15 N dipolar coupling measured using solid-state NMR methods w26x. It may be mentioned here that the isotropic 15 N chemical shift frequency of the Phe residue in polypeptides in solution ranges from 110.5 to 131.5 ppm with an average of 120.7 ppm. Also, solid-state NMR studies on n-acetylw1-13 CxGly-Lw15 Nx-PheNH 2 powder sample predicted three different 15 N isotropic chemical values Ž119, 121.4 and 123.3 ppm. from three magnetically inequivalent lattice environments w23,24x. These reported values are in good agreement with the value, 119 ppm, measured from the CPMAS spectrum in Fig. 1C. The possible sources of experimental errors of tensor values reported in this Letter could be the presence of multiple peptide conformations, molecular vibrations, and a variation Ž"0.02. in the scaling factor of the Lee–Goldburg decoupling sequence. It is worth pointing out that the magnitudes of the 15 N chemical shift tensors of 15 N-Phe-16 and 15 NGly-18 residues in the same magainin2 peptide are significantly different while the orientation of the tensors is the same within the experimental errors.
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D.K. Lee et al.r Chemical Physics Letters 309 (1999) 209–214
Fig. 2. Variation of the frequency of the shoulders in the simulated dipolar-shift spectrum of a powder sample of w15 N-Phe-16x-magainin2 peptide with the values of: ŽA. b N and ŽB. a N angles. Experimentally determined frequency values are shown in solid dots with an error bar.
The tensor parameters determined for the 15 N-Gly-18 residue are: s 11N s 42 " 2 ppm, s 22N s 73 " 2 ppm,
s 33N s 215 " 2 ppm, a N s 30 " 108 and b N s 22 " 28. Since the backbone conformation of the maga-
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inin2 peptide can be assumed to be the same in the sites of these two residues, the difference in the tensors could be attributed to the difference in the electronic environments at the 15 N nuclei in these residues. The similarity of a N values for the 15 N chemical shift tensors at Phe-16 and Gly-18 sites of the magainin2 peptide further confirms the theoretical prediction that the angle a N mostly depends on the dihedral angle c . The 15 N CSA tensors reported in this Letter are also significantly different from those determined from the amide sites of model peptides or small peptides in solids w10x. Therefore, care must be taken in the determination of the molecular backbone conformation of uniaxially oriented polypeptides, characterization of molecular backbone dynamics, and analysis of relaxation data using CSA tensors obtained from model peptides. Since it is not possible to characterize the spin interaction tensors of all amide sites in a protein, it would be fruitful to generate a large collection of experimental data in order to show the variation of CSA tensors on the primary and the secondary structures of polypeptides for the structural studies on proteins.
4. Conclusions Rigid 15 N chemical shift and 1 H– 15 N dipolar coupling tensors of 15 N-Phe residue in a powder sample of the magainin2 peptide have been successfully characterized using a simple one-dimensional dipolar-shift solid-state NMR method. The 15 N CSA tensor is significantly different from that of other amino acid residues in polypeptides. Based on the theoretical calculations and as suggested by the experimental results in this Letter, the deviation of the s 11N axis from the peptide plane and the s 22N axis from the normal to the peptide plane depends on the dihedral angle c of the peptide. These results should be useful in the structure determination of uniaxially oriented membrane-bound polypeptides or proteins using solid-state NMR spectroscopy w27,28x, to study partially aligned bicelles w29x using high-resolution solution NMR methods, and to examine the cross correlation studies in globular proteins w2–9,30x.
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Acknowledgements Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. This research was supported in part by the research funds from the Horace H. Rackham School of Graduate Studies and Michigan memorial Phoenix project at the University of Michigan. Funding support from the Cambridge Isotope Laboratories is also acknowledged.
References w1x W.P. Power, R.E. Wasylishen, Annu. Rep. NMR Spectrosc. 23 Ž1991. 1. w2x A. Shoji, S. Ando, S. Kuroki, I. Ando, G.A. Webb, Annu. Rep. NMR Spectrosc. 26 Ž1993. 55. w3x N. Tjandra, A. Szabo, A. Bax, J. Am. Chem. Soc. 118 Ž1996. 6986. w4x N. Tjandra, A. Bax, J. Am. Chem. Soc. 119 Ž1997. 9576. w5x K. Pervushin, R. Riek, G. Wider, K. Wuthrich, Proc. Natl. Acad. Sci. USA 94 Ž1997. 12366. w6x M.W.F. Fischer, L. Zeng, Y.X. Pang, W.D. Hu, A. Majumdar, E.R.P. Zuiderweg, J. Am. Chem. Soc. 119 Ž1997. 12629. w7x J. Boyd, C. Redfield, J. Am. Chem. Soc. 120 Ž1998. 9692. w8x D. Fushman, N. Tjandra, D. Cowburn, J. Am. Chem. Soc. 120 Ž1998. 10947. w9x D. Fushman, D. Cowburn, J. Biomol. NMR 13 Ž1999. 139. w10x D.K. Lee, R.J. Wittebort, A. Ramamoorthy, J. Am. Chem. Soc. 120 Ž1998. 8868, and references cited therein. w11x D.K. Lee, A. Ramamoorthy, J. Phys. Chem. B 103 Ž1999. 271. w12x C.H. Wu, A. Ramamoorthy, L.M. Gierasch, S.J. Opella, J. Am. Chem. Soc. 117 Ž1995. 6148. w13x D.K. Lee, A. Ramamoorthy, J. Magn. Reson. 133 Ž1998. 204. w14x A.E. Walling, R.E. Pargas, A.C. deDios, J. Phys. Chem. A 101 Ž1997. 7299. w15x A. Ramamoorthy, F.M. Marassi, M. Zasloff, S.J. Opella, J. Biomol. NMR 6 Ž1995. 329. w16x J. Gesell, M. Zasloff, S.J. Opella, J. Biomol. NMR 9 Ž1997. 127. w17x A.E. Bennett, C.M. Rienstra, M. Auger, K.V. Lakshmi, R.G. Griffin, J. Chem. Phys. 103 Ž1995. 6951. w18x G.S. Harbison, L.W. Jelinski, R.E. Stark, D.A. Torchia, J. Herzfeld, R.G. Griffin, J. Magn. Reson. 60 Ž1984. 79. w19x M. Munowitz, R.G. Griffin, G. Bodenhausen, T.H. Huang, J. Am. Chem. Soc. 103 Ž1980. 2529. w20x J.E. Roberts, G.S. Harbison, M.G. Munowitz, J. Herzfeld, R.G. Griffin, J. Am. Chem. Soc. 109 Ž1987. 4163.
214
D.K. Lee et al.r Chemical Physics Letters 309 (1999) 209–214
w21x Y. Hiyama, C.H. Niu, J.V. Silverton, A. Bavoso, D.A. Torchia, J. Am. Chem. Soc. 110 Ž1988. 2378. w22x W. Mai, W. Hu, T.A. Cross, Protein Sci. 2 Ž1993. 532. w23x T.G. Oas, C.J. Hartzell, F.W. Dahlquist, G. Drobny, J. Am. Chem. Soc. 109 Ž1987. 5962. w24x C.J. Hartzell, M. Whitfield, T.G. Oas, G.P. Drobny, J. Am. Chem. Soc. 109 Ž1987. 5966. w25x A. Kvick, A.R. Al-Karaghouli, T.F. Koetzle, Acta Crystallogr. B 33 Ž1977. 3796.
w26x T. Nakai, J. Ashida, T. Terao, Mol. Phys. 67 Ž1989. 839. w27x T.A. Cross, S.J. Opella, Curr. Opin. Struct. Biol. 4 Ž1994. 574. w28x D.E. Warschawski, M. Traikia, P.F. Devaux, G. Bodenhausen, Biochimie 80 Ž1998. 437. w29x C.R. Sanders, B.J. Hare, K.P. Howard, J. Prestegard, Prog. NMR Spectrosc. 26 Ž1993. 421. w30x D.W. Yang, A. Mittermaier, Y.K. Mok, L.E. Kay, J. Mol. Biol. 276 Ž1998. 939.