Abstract
A constant-time, Carr–Purcell–Meiboom–Gill (CPMG) transverse relaxation, R2, dispersion experiment for carbonyl carbons was designed and executed to detect μs-ms time-scale dynamics of protein backbone carbonyl sites. Because of the large (ca. 55 Hz) Cα-C′ J-coupling, the carbonyl signal intensity is strongly modulated as the spacing between CPMG pulses is varied, in uniformly 13C enriched proteins, unless care is taken to minimize the perturbation of the Cα magnetization by the CPMG pulses. CPMG pulse trains consisting of either a band-selective pulse, such as RE-BURP, or rectangular (with an excitation null in the Cα region of the spectrum) pulses were employed in order to minimize C′ signal modulation by Cα-C′ J-coupling. The performance of these types of CPMG refocusing pulses was assessed by computer simulation, and by comparing dispersion profiles measured for (1) uniformly [13C,15N, 2H] (2H at non-labile hydrogen sites) labeled, and (2) uniformly 15N/selectively-13C′ labeled samples of HIV-1 protease bound to a potent inhibitor, DMP323. In addition, because the uniformly 13C/15N/2H labeled sample was well suited to measure 15N and 1H R2 dispersion as well as 13C′ dispersion, conformational exchange in the inter subunit β-sheet hydrogen-bond network of the inhibitor-bound protease was elucidated using relaxation dispersion data of all three types of nuclei.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Cavanagh, J., Fairbrother, W.J., Palmer, r., A.G. and Skelton, N.J. (1996) Protein NMR Spectroscopy, Academic Press, San Diego, pp. 135.
Cornilescu, G. and Bax, A. (2000) J. Am. Chem. Soc., 122, 10143–10154.
Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) J. Biomol. NMR, 6, 277–293.
Ernst, R.R., Bodenhausen, G. and Wokaun, A. (1987) Principles of Nuclear Magnetic Resonance in One and Two Dimensions, Clarendon Press, Oxford.
Garrett, D.S., Powers, R., Gronenborn, A.M. and Clore, G.M. (1991) J. Magn. Reson., 95, 214–220.
Geen, H. and Freeman, R. (1991) J. Mag. Reson., 93, 93–141.
Hu, J.S. and Bax, A. (1996) J. Am. Chem. Soc., 118, 8170–8171.
Hu, J.S., Grzesiek, S. and Bax, A. (1997) J. Am. Chem. Soc., 119, 1803–1804.
Ishima, R., Freedberg, D.I., Wang, Y.X., Louis, J.M. and Torchia, D.A. (1999) Structure, 7, 1047–1055.
Ishima, R., Louis, J.M. and Torchia, D.A. (2001) J. Mol. Biol., 305, 515–521.
Ishima, R. and Torchia, D.A. (2003) J. Biomol. NMR, 25, 243–348.
Ishima, R., Wingfield, P.T., Stahl, S.J., Kaufman, J.D. and Torchia, D.A. (1998) J. Am. Chem. Soc., 120, 10534–10542.
Katoh, E., Louis, J.M., Yamazaki, T., Gronenborn, A.M., Torchia, D.A. and Ishima, R. (2003) Protein Sci., 12, 1376–1385.
Loria, J.P., Rance, M. and Palmer, A.G. (1999) J. Am. Chem. Soc., 121, 2331–2332.
Louis, J.M., Ishima, R., Nesheiwat, I., Pannell, L.K., Lynch, S.M., Torchia, D.A. and Gronenborn, A.M. (2003) J. Biol. Chem., 278, 6085–6092.
Marion, D., Ikura, M., Tschudin, R. and Bax, A. (1989) J. Magn. Reson., 85, 393–399.
Matsuo, H., Kupce, E., Li, H.J. and Wagner, G. (1996) J. Magn. Reson. Ser. B., 113, 91–96.
Millet, O., Loria, J.P., Kroenke C.D. and Palmer A.G. (2000) J. Am. Chem. Soc., 122, 2867–2877.
Mulder, F.A.A. and Akke, M. (2003) Magn. Reson. Chem., 41, 853–865.
Mulder, F.A.A., Hon, B., Mittermaier, A., Dahlquist, F.W. and Kay, L.E. (2002) J. Am. Chem. Soc., 124, 1443–1451.
Mulder, F.A.A., Skrynnikov, N.R., Hon, B., Dahlquist, F.W. and Kay, L.E. (2001) J. Am. Chem. Soc., 123, 967–975.
Neal, S., Nip, A.M., Zhang, H. and Wishart, D.S. (2003) J. Biomol. NMR, 26, 215–240.
Palmer, A.G., Kroenke, C.D. and Loria, J.P. (2001) Meth. Enzymol., 339, 204–238.
Skrynnikov, N.R., Dahlquist, F.W. and Kay, L.E. (2002) J. Am. Chem. Soc., 124, 12352–12360.
Skrynnikov, N.R., Mulder, F.A.A., Hon, B., Dahlquist, F.W. and Kay, L.E. (2001) J. Am. Chem. Soc., 123, 4556–4566.
Stryer, L. (1995) In Biochemistry, W.H. Freeman and Company, New York, pp. 713–738.
Tollinger, M., Skrynnikov, N.R., Mulder, F.A.A., Forman-Kay, J.D. and Kay, L.E. (2001) J. Am. Chem. Soc., 123, 11341–11352.
Yamazaki, T., Hinck, A.P., Wang, Y.X., Nicholson, L.K., Torchia, D.A., Wingfield, P., Stahl, S.J., Kaufman, J.D., Chang, C.H., Domaille, P.J. and Lam, P.Y. (1996) Protein Sci., 5, 495–506.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ishima, R., Baber, J., Louis, J.M. et al. Carbonyl carbon transverse relaxation dispersion measurements and ms-μs timescale motion in a protein hydrogen bond network. J Biomol NMR 29, 187–198 (2004). https://doi.org/10.1023/B:JNMR.0000019249.50306.5d
Issue Date:
DOI: https://doi.org/10.1023/B:JNMR.0000019249.50306.5d