Abstract
Three solution NMR experiments on a uniformly 15N labeled membrane protein in micelles provide sufficient information to describe the structure, topology, and dynamics of its helices, as well as additional information that characterizes the principal features of residues in terminal and inter-helical loop regions. The backbone amide resonances are assigned with an HMQC-NOESY experiment and the backbone dynamics are characterized by a 1H-15N heteronuclear NOE experiment, which clearly distinguishes between the structured helical residues and the more mobile residues in the terminal and interhelical loop regions of the protein. The structure and topology of the helices are described by Dipolar waves and PISA wheels derived from experimental measurements of residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). The results show that the membrane-bound form of Pf1 coat protein has a 20-residue trans-membrane hydrophobic helix with an orientation that differs by about 90° from that of an 8-residue amphipathic helix. This combination of three-experiments that yields Dipolar waves and PISA wheels has the potential to contribute to high-throughput structural characterizations of membrane proteins.
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Almeida, F.C.L. and Opella, S.J. (1997) J. Mol. Biol., 270, 481–495.
Bax, A., Kontaxis, G. and Tjandra, N. (2001) Meth. Enzymol., 339, 127–174.
Chou, J.J., Gaemers, S., Howder, B., Luis, J.M. and Bax, A. (2001) J. Biomol. NMR., 21, 377–382.
Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) J. Biomol. NMR, 6, 277–293.
Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Biochemistry, 33, 5984–6003.
Ishii, Y., Markus, M.A. and Tycko, R. (2001) J. Biomol. NMR, 21, 141–151.
Ketchem, R.R., Hu, W. and Cross, T.A. (1993) Science, 261, 1457–1460.
Koradi, R., Billeter, M. and Wüthrich, K. (1996) J. Mol. Graph., 14, 51–55.
Ma, C. and Opella, S.J. (2000) J. Magn. Reson., 146, 381–384.
Marassi, F.M. and Opella, S.J. (2000) J. Magn. Reson., 144, 150–155.
Marassi, F.M. and Opella, S.J. (2003) Protein Sci., 12, 403–411.
Mesleh, M.F., Veglia, G., DeSilva, T.M., Marassi, F.M. and Opella, S.J. (2002) J. Am. Chem. Soc., 22, 4206–4207.
Mori S., Abeygunawardana, C., Johnson, M.O. and van Zijl, P.C.M. (1995) J. Magn. Reson. B, 108, 94–98.
Opella, S.J., Marassi, F.M., Gesell, J.J., Valente, A.P., Kim, Y., Oblatt-Montal, M. and Montal, M. (1999) Nat. Struct. Biol., 6, 374–379.
Opella, S.J., Nevzorov, A., Mesleh, M.F. and Marassi, F.M. (2002) Biochem. Cell Biol., 80, 597–604.
Opella, S.J., Stewart, P.L. and Valentine, K.G. (1987) Quart. Rev. Biophys., 19, 7–49.
Ottiger, M., Delaglio, F. and Bax, A. (1998) J. Magn. Reson., 131, 373–378.
Prestegard J.H., Valafar, H., Glushka, J. and Tian, F. (2001) Biochemistry, 40, 8677–8685.
Sass, H.J., Musco, G., Stahl, S.J., Wingfield, P.T. and Grzesiek, S. (2000) J. Biomol. NMR, 18, 303–309.
Schiksnis R.A., Bogusky, M.J., Tsang, P. and Opella, S.J. (1987) Biochemistry, 26, 1373–1381.
Schwieters, C.D., Kuszewski, J.J., Tjandra, N. and Clore, G.M. (2003) J. Magn. Reson., 160, 65–73.
Shon, K.-J. and Opella, S.J. (1989) J. Magn. Reson., 82, 193–197.
Shon, K.-J., Kim, Y., Colnago, L.A. and Opella, S.J. (1991) Science, 252, 1303–1305.
Tobias, D.J., Gesell, J., Klein, M.L. and Opella, S.J. (1995) J. Mol. Biol., 253, 391–395.
Tycko, R., Blanco, F.J. and Ishii, Y. (2000) J. Am. Chem. Soc., 122, 9340–9341.
Veglia, G. and Opella, S.J. (2000) J. Am. Chem. Soc., 122, 11733–11734.
Wang J., Denny, J., Tian, C., Kim, S., Mo, Y., Kovacs, F., Song, Z., Nishimura, K., Gan, Z., Fu, R., Quine, J.R. and Cross, T.A. (2000) J. Magn. Reson., 144, 162–167.
Wang, J., Kim, S., Kovacs, F. and Cross, T.A. (2001) Protein Sci., 10, 2241–2250.
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Lee, S., Mesleh, M.F. & Opella, S.J. Structure and dynamics of a membrane protein in micelles from three solution NMR experiments. J Biomol NMR 26, 327–334 (2003). https://doi.org/10.1023/A:1024047805043
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DOI: https://doi.org/10.1023/A:1024047805043