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Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins

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Abstract

NMR spin relaxation in the rotating frame (R1ρ) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide 1H off-resonance R1ρ relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using 15N or 13C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The 1H off-resonance R1ρ data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 μs. This is in contrast to the case for 15N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly 15N-enriched samples that were either highly enriched in 2H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the 1H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present 1H off-resonance R1ρ experiments are in excellent agreement with those determined previously using a combination of 15N laboratory-frame and off-resonance R1ρ relaxation data, with average values of \(\left\langle {\tau _{{\rm{ex}}} } \right\rangle {\rm{ = 19}} \pm {\rm{7}}\)and 21 ± 3 μs, respectively.

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Abbreviations

DSS:

2,2-dimethyl-2-silapentane-5-sulfonic acid

Tr2C:

the C-terminal domain of bovine calmodulin (residues 76–148).

References

  • M. Akke (2002) Curr. Opin. Struct. Biol. 12 642–647 Occurrence Handle10.1016/S0959-440X(02)00369-X Occurrence Handle12464317

    Article  PubMed  Google Scholar 

  • M. Akke A.G. Palmer (1996) J. Am. Chem. Soc. 118 911–912 Occurrence Handle10.1021/ja953503r

    Article  Google Scholar 

  • F.C.L. Almeida S.J. Opella (1997) J. Magn. Reson. 124 509–511 Occurrence Handle10.1006/jmre.1996.1091 Occurrence Handle9169225

    Article  PubMed  Google Scholar 

  • Cavanagh, J., Fairbrother, W.J., Palmer, A.G. and Skelton, N.J., (1995) Protein NMR Spectroscopy: Principles and Practice, Academic Press, San Diego

  • R. Chattopadhyaya W.E. Meador A.R. Means F.A. Quiocho (1992) J. Mol. Biol. 228 1177–1192 Occurrence Handle10.1016/0022-2836(92)90324-D Occurrence Handle1474585

    Article  PubMed  Google Scholar 

  • J.J. Chou S.P. Li C.B. Klee A. Bax (2001) Nat. Struct. Biol. 8 990–997 Occurrence Handle10.1038/nsb1101-990 Occurrence Handle11685248

    Article  PubMed  Google Scholar 

  • A. Crivici M. Ikura (1995) Ann. Rev. Biophys. Biomol. Struct. 24 85–116 Occurrence Handle10.1146/annurev.bb.24.060195.000505

    Article  Google Scholar 

  • D.G. Davis M.E. Perlman R.E. London (1994) J. Magn. Reson. B104 266–275

    Google Scholar 

  • F. Delaglio S. Grzesiek G.W. Vuister G. Zhu J. Pfeifer A. Bax (1995) J. Biomol. NMR 6 277–293 Occurrence Handle10.1007/BF00197809 Occurrence Handle8520220

    Article  PubMed  Google Scholar 

  • H. Desvaux P. Berthault N. Birlirakis M. Goldman (1994) J. Magn. Reson. A108 219–229

    Google Scholar 

  • H. Desvaux N. Birlirakis C. Wary P. Berthault (1995) Mol. Phys. 86 1059–1073

    Google Scholar 

  • Devore J.L., (1999) Probability and Statistics for Engineering and the Sciences, Brooks/Cole Publishing Company, Monterey

  • E.Z. Eisenmesser D.A. Bosco M. Akke D. Kern (2002) Science 295 1520–1523 Occurrence Handle10.1126/science.1066176 Occurrence Handle11859194

    Article  PubMed  Google Scholar 

  • J. Evenäs S. Forsén A. Malmendal M. Akke (1999) J. Mol. Biol. 289 603–617 Occurrence Handle10.1006/jmbi.1999.2770 Occurrence Handle10356332

    Article  PubMed  Google Scholar 

  • J. Evenäs A. Malmendal M. Akke (2001) Structure 9 185–195 Occurrence Handle10.1016/S0969-2126(01)00575-5 Occurrence Handle11286885

    Article  PubMed  Google Scholar 

  • J. Evenäs E. Thulin A. Malmendal S. Forsén G. Carlström (1997) Biochemistry 36 3448–3457 Occurrence Handle10.1021/bi9628275 Occurrence Handle9131994

    Article  PubMed  Google Scholar 

  • B.E. Finn J. Evenäs T. Drakenberg J.P. Waltho E. Thulin S. Forsén (1995) Nat. Struct. Biol. 2 777–783 Occurrence Handle10.1038/nsb0995-777 Occurrence Handle7552749

    Article  PubMed  Google Scholar 

  • M. Garwood Y. Ke (1991) J. Magn. Reson. 94 511–525

    Google Scholar 

  • H. Geen R. Freeman (1991) J. Magn. Reson. 93 93–141

    Google Scholar 

  • M.J. Grey C. Wang A.G. Palmer (2003) J. Am. Chem. Soc. 125 14324–14335 Occurrence Handle10.1021/ja0367389 Occurrence Handle14624581

    Article  PubMed  Google Scholar 

  • R. Ishima P.T. Wingfield S.J. Stahl J.D. Kaufman D.A. Torchia (1998) J. Am. Chem. Soc. 120 10534–10542 Occurrence Handle10.1021/ja981546c

    Article  Google Scholar 

  • L.E. Kay L.K. Nicholson F. Delagio A. Bax D.A. Torchia (1992) J. Magn. Reson. 97 359–375

    Google Scholar 

  • P.A. Keifer (1999) Conc. Magn. Reson. 11 165–180 Occurrence Handle10.1002/(SICI)1099-0534(1999)11:3<165::AID-CMR4>3.0.CO;2-D

    Article  Google Scholar 

  • J.G. Kempf J.Y. Jung N.S. Sampson J.P. Loria (2003) J.Am. Chem. Soc. 124 12064–12065 Occurrence Handle10.1021/ja037101s

    Article  Google Scholar 

  • D.M. Korzhnev V.Y. Orekhov L.E. Kay (2005) J. Am. Chem. Soc. 127 713–721 Occurrence Handle10.1021/ja0446855 Occurrence Handle15643897

    Article  PubMed  Google Scholar 

  • D.M. Korzhnev X. Salvatella M. Vendruscolo A.A. Di Nardo A. R. Davidson C.M. Dobson L.E. Kay (2004) Nature 430 586–590 Occurrence Handle10.1038/nature02655 Occurrence Handle15282609

    Article  PubMed  Google Scholar 

  • D.M. Korzhnev N.R. Skrynnikov O. Millet D.A. Torchia L.E. Kay (2002) J. Am. Chem. Soc. 124 10743–10753 Occurrence Handle10.1021/ja0204776 Occurrence Handle12207529

    Article  PubMed  Google Scholar 

  • C.D. Kroenke J.P. Loria L.K. Lee M. Rance A.G. Palmer (1998) J. Am. Chem. Soc. 120 7905–7915 Occurrence Handle10.1021/ja980832l

    Article  Google Scholar 

  • H. Kuboniwa N. Tjandra S. Grzesiek H. Ren C.B. Klee A. Bax (1995) Nat. Struct. Biol. 2 768–776 Occurrence Handle10.1038/nsb0995-768 Occurrence Handle7552748

    Article  PubMed  Google Scholar 

  • H. Le E. Oldfield (1994) J. Biomol. NMR 4 341–348 Occurrence Handle10.1007/BF00179345 Occurrence Handle8019141

    Article  PubMed  Google Scholar 

  • P. Lundström M. Akke (2004) J. Am. Chem. Soc. 126 928–935 Occurrence Handle10.1021/ja037529r Occurrence Handle14733570

    Article  PubMed  Google Scholar 

  • A. Malmendal J. Evenäs S. Forsén M. Akke (1999) J. Mol. Biol. 293 883–899 Occurrence Handle10.1006/jmbi.1999.3188 Occurrence Handle10543974

    Article  PubMed  Google Scholar 

  • D. Marion M. Ikura R. Tschudin A. Bax (1989) J. Magn. Reson. 85 393–399

    Google Scholar 

  • F. Massi E. Johnson C. Wang M. Rance A.G. Palmer (2004) J. Am. Chem. Soc. 126 2247–2258 Occurrence Handle10.1021/ja038721w Occurrence Handle14971961

    Article  PubMed  Google Scholar 

  • F.A.A. Mulder M. Akke (2003) Magn. Reson. Chem. 41 853–865 Occurrence Handle10.1002/mrc.1252

    Article  Google Scholar 

  • F.A.A. Mulder R.A. Graaf Particlede R. Kaptein R. Boelens (1998) J. Magn. Reson. 131 351–357 Occurrence Handle10.1006/jmre.1998.1380 Occurrence Handle9571112

    Article  PubMed  Google Scholar 

  • F.A.A. Mulder B. Hon A. Mittermaier F.W. Dahlquist L.E. Kay (2002) J. Am. Chem. Soc. 124 1443–1451 Occurrence Handle10.1021/ja0119806 Occurrence Handle11841314

    Article  PubMed  Google Scholar 

  • M. Ottiger F. Delaglio A. Bax (1998) J. Magn. Reson. 131 373–378 Occurrence Handle10.1006/jmre.1998.1361 Occurrence Handle9571116

    Article  PubMed  Google Scholar 

  • A.G. Palmer (2004) Chem. Rev. 104 3623–3640 Occurrence Handle10.1021/cr030413t Occurrence Handle15303831

    Article  PubMed  Google Scholar 

  • A.G. Palmer C.D. Kroenke J.P. Loria (2001) Methods Enzymol. 339 204–238 Occurrence Handle11462813

    PubMed  Google Scholar 

  • A.G. Palmer N.J. Skelton W.J. Chazin P.E. Wright M. Rance (1992) Mol. Phys. 75 699–711

    Google Scholar 

  • K. Pervushin R. Riek G. Wider K. Wüthrich (1997) Proc. Natl. Acad. Sci. USA 94 12366–12371 Occurrence Handle10.1073/pnas.94.23.12366 Occurrence Handle9356455

    Article  PubMed  Google Scholar 

  • Press W.H., Flannery B.P., Teukolsky S.A. and Vetterling W.T. (1988) Numerical Recipes in C. The Art of Scientific Computing Cambridge University Press Cambridge

  • A.J. Shaka J. Keeler R. Freeman (1983) J. Magn. Reson. 53 313–340

    Google Scholar 

  • V. Sklenar M. Piotto R. Leppik V. Saudek (1993) J.␣Magn. Reson. A102 241–245 Occurrence Handle10.1006/jmra.1993.1098

    Article  Google Scholar 

  • O. Trott D. Abergel A.G. Palmer (2003) Mol. Phys. 101 753–763 Occurrence Handle10.1080/0026897021000054826

    Article  Google Scholar 

  • O. Trott A.G. Palmer (2002) J. Magn. Reson. 154 157–160 Occurrence Handle10.1006/jmre.2001.2466 Occurrence Handle11820837

    Article  PubMed  Google Scholar 

  • K. Ugurbil M. Garwood A.R. Rath (1988) J. Magn. Reson. 80 448–469

    Google Scholar 

  • van de Ven, F.J.M. (1995) Multidimensional NMR in liquids. Basic Principles and Experimental Methods Wiley-VCH, New York

  • H.J. Vogel (1994) Biochem. Cell Biol. 72 357–376 Occurrence Handle7605608

    PubMed  Google Scholar 

  • A.C. Wang A. Bax (1993) J. Biomol. NMR 3 715–720 Occurrence Handle10.1007/BF00198374 Occurrence Handle8111234

    Article  PubMed  Google Scholar 

  • C.Y. Wang M.J. Grey A.G. Palmer (2001) J. Biomol. NMR 21 361–366 Occurrence Handle10.1023/A:1013328206498 Occurrence Handle11824755

    Article  PubMed  Google Scholar 

  • D.S. Wishart D.A. Case (2001) Methods Enzymol. 338 3–34 Occurrence Handle11460554

    PubMed  Google Scholar 

  • M. Wolf-Watz V. Thai K. Henzler-Wildman G. Hadjipavlou E. Z. Eisenmesser D. Kern (2004) Nat. Struct. Mol. Biol. 11 945–949 Occurrence Handle10.1038/nsmb821 Occurrence Handle15334070

    Article  PubMed  Google Scholar 

  • X.P. Xu D.A. Case (2002) Biopolymers 65 408–423 Occurrence Handle10.1002/bip.10276 Occurrence Handle12434429

    Article  PubMed  Google Scholar 

  • J. Yan Y. Liu S.M. Lukasik N.A. Speck J.H. Bushweller (2004) Nat. Struct. Mol. Biol. 11 901–906 Occurrence Handle10.1038/nsmb819 Occurrence Handle15322525

    Article  PubMed  Google Scholar 

  • M. Zhang T. Tanaka M. Ikura (1995) Nat. Struct. Biol. 2 758–767 Occurrence Handle10.1038/nsb0995-758 Occurrence Handle7552747

    Article  PubMed  Google Scholar 

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  1. Department of Biophysical Chemistry, Lund University, P.O. Box 124, SE-22100, Lund, Sweden

    Patrik Lundström & Mikael Akke

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  1. Patrik Lundström
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Lundström, P., Akke, M. Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins. J Biomol NMR 32, 163–173 (2005). https://doi.org/10.1007/s10858-005-5027-3

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