Skip to main content
Log in

Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

An Erratum to this article was published on 01 March 2007

Abstract

A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring 15N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone 15N R 1, R , 15N–{1H} NOE, and 15N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsATM, exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, τc = 38 ± 2.5 ns, at 50 °C and a diffusion anisotropy, \({D}_{\parallel}/{D}_{\bot}=0.79\pm 0.05\), corresponding to an aspect ratio a/b  ≥  1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S 2 values in the 0.2–0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps–ns time scale for the 5-residue selectivity filter, which selects K+ ions to enter the channel.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Akke M. (2002) NMR methods for characterizing microsecond to millisecond dynamics in recognition and catalysis. Curr. Opin. Struct. Biol. 12: 642–647

    Article  Google Scholar 

  • Arora A., Tamm L.K. (2001) Biophysical approaches to membrane protein structure determination. Curr. Opin. Struct. Biol. 11: 540–547

    Article  Google Scholar 

  • Bruschweiler R. (2003) New approaches to the dynamic interpretation and prediction of NMR relaxation data from proteins. Curr. Opin. Sruct. Biol. 13: 175–183

    Article  Google Scholar 

  • Buevich A.V., Shinde U.P., Inouye M., Baum J. (2001) Backbone dynamics of the natively unfolded pro-peptide of subtilisin by heteronuclear NMR relaxation studies. J. Biomol. NMR 20: 233–249

    Article  Google Scholar 

  • Caffrey M., Kaufman J., Stahl S.J., Wingfield P.T., Gronenborn A.M., Clore G.M. (1998) 3D NMR experiments for measuring 15N relaxation data of large proteins: application to the 44 kDa ectodomain of SIV gp41. J. Magn. Res. 135: 368–372

    Article  ADS  Google Scholar 

  • Cantor, C.R. and Schimmel, P.R. (1980). Biophysical Chemistry, vol. 2, W.H. Freeman and Company, New York. pp. 562–565

  • Carr P.A., Fearing D.A., Palmer A.G.I. (1998) 3D-accordion spectroscopy for measuring 15N and 13CO relaxation rates in poorly resolved NMR spectra. J. Magn. Res. 132: 25–33

    Article  ADS  Google Scholar 

  • Chill J.H., Louis J.M., Miller C., Bax A. (2006) NMR study of the tetrameric KcsA potassium channel in detergent micelles. Prot. Sci. 15: 684–698

    Article  Google Scholar 

  • Choi H., Heginbotham L. (2004) Functional influence of the pore helix glutamate in the KcsA K+ channel. Biophys. J. 86: 2137–2144

    Article  Google Scholar 

  • Clore G.M., Driscoll P.C., Wingfield P.T., Gronenborn A.M. (1990) Analysis of backbone dynamics of interleukin-1-beta using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. Biochemistry 29: 7387–7401

    Article  Google Scholar 

  • Cole R., Loria J.P. (2003) FAST-Modelfree: a program for rapid automated analysis of solution NMR spin-relaxation data. J. Biomol. NMR 13: 289–302

    Google Scholar 

  • Copie V., Tomita Y., Akiyama S.K., Aota S., Yamada K.M., Venable R.M., Pastor R.W., Krueger S., Torchia D.A. (1998) Solution structure and dynamics of linked cell attachment modules of mouse fibronectin containing the RGD and synergy regions: comparison with the human fibronectin crystal structure. J. Mol. Biol. 277: 663–82

    Article  Google Scholar 

  • Cornilescu G., Bax A. (2000) Measurement of proton, nitrogen and carbonyl chemical shielding anisotropies in a protein dissolved in a dilute liquid crystalline phase. J. Am. Chem. Soc. 122: 10143–10154

    Article  Google Scholar 

  • Cortes D.M., Cuello L.G., Perozo E. (2001) Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating. J. Gen. Physiol. 117: 165–180

    Article  Google Scholar 

  • Cuello L.G., Romero J.G., Cortes D.M., Perozo E. (1998) pH-dependent gating in the Streptomyces lividans K+ channel. Biochemistry 37: 3229–3236

    Article  Google Scholar 

  • Delaglio F., Grzesiek S., Vuister G.W., Zhu G., Pfeifer J., Bax A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6: 277–293

    Article  Google Scholar 

  • Doyle D.A., Cabral J.M., Pfuetzner R.A., Kuo A., Gulbis J.M., Cohen S.L., Chait B.T., Mackinnon R. (1998) The structure of the potassium channel: Molecular basis of K+ conduction and selectivity. Science 280: 69–77

    Article  ADS  Google Scholar 

  • Eisenmesser E.Z., Bosco D.A., Akke M., Kern D. (2002) Enzyme dynamics during catalysis. Science 295: 1520–1523

    Article  ADS  Google Scholar 

  • 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., Kay L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33: 5984–6003

    Article  Google Scholar 

  • Farrow N.A., Zhang O., Szabo A., Torchia D.A., Kay L.E. (1995) Spectral density function mapping using 15N relaxation data exclusively. J. Biomol. NMR 6: 153–162

    Article  Google Scholar 

  • Ghose R., Eykyn T.R., Bodenhausen G. (1999) Average Liouvillian theory revisited: cross-correlated relaxation between chemical shift anisotropy and dipolar couplings in the rotating frame in nuclear magnetic resonance. Mol. Phys. 96: 1281–1288

    Article  ADS  Google Scholar 

  • Goldman M. (1984) Interference effects in the relaxation of a pair of unlike spin-1/2 nuclei. J. Magn. Res. 60: 437–452

    Google Scholar 

  • Grey M.J., Wang C., Palmer A.G. III (2003) Disulfide bond isomerization in basic pancreatic trypsin inhibitor: multisite chemical exchange quantified by CPMG relaxation dispersion and chemical shift modeling. J. Am. Chem. Soc. 125: 14324–14335

    Article  Google Scholar 

  • Hardy R.C., Cottington R.L. (1949) Viscosity of deuterium oxide and water from 5 to 125 °C. J. Chem. Phys. 17: 509–510

    Article  ADS  Google Scholar 

  • Heginbotham L., LeMasurier M., Kolmakova-Partensky L., Miller C. (1999) Single streptomyces lividans K+ channels: functional asymmetries sidedness of proton activation. J. Gen. Physiol. 114: 551–560

    Article  Google Scholar 

  • Ishima R., Torchia D.A. (2000) Protein dynamics from NMR. Nat. Struct. Biol. 7: 740–743

    Article  Google Scholar 

  • Jones J.A., Hodgkinson R., Barker A.L., Hore P.J. (1996) Optimal sampling strategies for the measurement of spin-spin relaxation times. J. Magn. Res. Ser B 113: 25–34

    Article  Google Scholar 

  • Kay L.E. (1998) Protein dynamics from NMR. Biochem. Cell Biol. 76: 145–152

    Article  Google Scholar 

  • Kay L.E., Torchia D.A., Bax A. (1989) Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy – application to staphylococcal nuclease. Biochemistry 28: 8972–8979

    Article  Google Scholar 

  • Kempf J.G., Loria J.P. (2003) Protein dynamics from solution NMR: theory and applications. Cell Biochem. Biophys. 37: 187–211

    Article  Google Scholar 

  • Kern D., Zuiderweg E.R. (2003) The role of dynamics in allosteric regulation. Curr. Opin. Sruct. Biol. 13: 748–757

    Article  Google Scholar 

  • Korzhnev D.M., Orekhov V.Y., Dahlquist F.W., Kay L.E. (2003) Off-resonance R relaxation outside of the fast exchange limit: an experimental study of a cavity mutant of T4 lysozyme. J. Biomol. NMR 26: 39–48

    Article  Google Scholar 

  • Kroenke C.D., Loria J.P., Lee L.K., Rance M., Palmer A.G. III (1998) Longitudinal and transverse 1H–15N dipolar chemical shift anisotropy relaxation interference: unambiguous determination of rotational diffusion tensors and chemical exchange effects in biological macromolecules. J. Am. Chem. Soc. 120: 7905–7915

    Article  Google Scholar 

  • Lee D., Hilty C., Wider G., Wuthrich K. (2006) Effective rotational correlation times of proteins from NMR relaxation interference. J. Magn. Res. 178: 72–76

    Article  ADS  Google Scholar 

  • Lee L.K., Rance M., Chazin W.J., Palmer A.G. III (1997) Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13Cα nuclear spin relaxation. J. Biomol. NMR 9: 287–298

    Article  Google Scholar 

  • Lipari G., Szabo A. (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. 2. Analysis of experimental results. J. Am. Chem. Soc. 104: 4546–4570

    Article  Google Scholar 

  • Lohr F., Katsemi V., Hartleib J., Gunther U., Ruterjanz H. (2003) A strategy to obtain backbone resonance assignments of deuterated proteins in the presence of incomplete amide 2H/1H back-exchange. J. Biomol. NMR 25: 291–311

    Article  Google Scholar 

  • Loria J.P., Rance M., Palmer A.G. III (1999) Transverse-relaxation-optimized (TROSY) gradient-enhanced triple-resonance NMR spectroscopy. J. Magn. Res. 141: 180–184

    Article  ADS  Google Scholar 

  • Mandel A.M., Akke M., Palmer A.G. III (1995) Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme. J. Mol. Biol. 246: 144–163

    Article  Google Scholar 

  • McCallum S.A., Hitchens T.K., Rule G.S. (1999) Solution structure of the carboxyl terminus of a human class μ-glutathione S-transferase: NMR assignment strategies in large proteins. J. Mol. Biol. 285: 2119–2132

    Article  Google Scholar 

  • Molina M.L., Encinar J.A., Barrera F.N., Fernandez-Ballester G., Riquelme G., Gonzalez-Ros J.M. (2004) Influence of C-terminal protein domains and protein–lipid interactions on tetramerization and stability of the potassium channel KcsA. Biochemistry 43: 14924–14931

    Article  Google Scholar 

  • Mulder F.A., de Graaf R.A., Kaptein R., Boelens R. (1998) An off-resonance rotating-frame experiment for the investigation of macromolecular dynamics using adiabatic rotations. J. Magn. Res. 131: 351–357

    Article  ADS  Google Scholar 

  • Mulder F.A., Mittermaier A., Hon B., Dahlquist F.W., Kay L.E. (2001) Studying excited states of proteins by NMR spectroscopy. Nat. Struct. Biol. 8: 932–935

    Article  Google Scholar 

  • Palmer A.G. III, Kroenke C.D., Loria J.P. (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Meth. Enzymol. 339: 204–238

    Article  Google Scholar 

  • Palmer A.G. III (2001) NMR probes of molecular dynamics: overview and comparison with other techniques. Annu. Rev. Biophys. Biomol. Struct. 30: 129–155

    Article  Google Scholar 

  • Peng J.W., Wagner G. (1992) Mapping of the spectral densities of N–H bond motions in eglin-C using heteronuclear relaxation measurements. Biochemistry 31: 8571–8586

    Article  Google Scholar 

  • Pervushin K., Wider G., Wuthrich K. (1998) Single transition-to-single transition polarization transfer (ST2-PT) in [15N,1H]-TROSY. J. Biomol. NMR 12: 345–348

    Article  Google Scholar 

  • Renner C., Moroder L., Holak T.A. (2001) Analytical solution to the Lipari-Szabo model based on the reduced spectral density approximation offers a novel protocol for extracting motional parameters. J. Magn. Res. 151: 32–39

    Article  ADS  Google Scholar 

  • Schurr J.M., Babcock H.P., Fujimoto B.S. (1994) A test of the model-free formulas – effects of anisotropic rotational diffusion an dimerization. J. Magn. Res. Ser B 105: 211–224

    Article  Google Scholar 

  • Schwalbe H., Fiebig K.M., Buck M., Jones J.A., Grimshaw S.B., Spencer A., Glaser S.J., Smith L.J., Dobson C.M. (1997) Structural and dynamical properties of a denatured protein. Heteronuclear 3D NMR experiments and theoretical simulations of lysozyme in 8 M urea. Biochemistry 36: 8977–8991

    Article  Google Scholar 

  • Schwarzinger S., Wright P.E., Dyson H.J. (2002) Molecular hinges in protein folding: the urea-denatured state of apomyoglobin. Biochemistry 41: 12681–12686

    Article  Google Scholar 

  • Silver M.S., Joseph R.I., Chen C.N., Sank V.J., Hoult D.I. (1984) Selective population inversion in NMR. Nature 310: 681–683

    Article  ADS  Google Scholar 

  • Tian C., Karra M.D., Ellis C.D., Jacob J., Oxenoid K., Sonnichsen F., Sanders C.R. (2005) Membrane protein preparation for TROSY NMR screening. Meth. Enzymol. 394: 321–334

    Article  Google Scholar 

  • Tjandra N., Szabo A., Bax A. (1996) Protein backbone dynamics and 15N chemical shift anisotropy from quantitative measurement of relaxation interference effects. J. Am. Chem. Soc. 118: 6986–6991

    Article  Google Scholar 

  • Tugarinov V., Ollerenshaw J.E., Kay L.E. (2005) Probing side-chain dynamics in high molecular weight proteins by deuterium NMR spin relaxation: an application to an 82-kDa enzyme. J. Am. Chem. Soc. 127: 8214–8225

    Article  Google Scholar 

  • Viles J.H., Duggan B.M., Zaborowski E., Schwarzinger S., Huntley J.J.A., Kroon G.J.A., Dyson H.J., Wright P.E. (2001) Potential bias in NMR relaxation data introduced by peak intensity analysis and curve fitting methods. J. Biomol. NMR 21: 1–9

    Article  Google Scholar 

  • Wagner G., Nirmala N.R. (1989) Studies of protein dynamics by heteronuclear NMR – individual 13C relaxation times and evidence for multiple conformations in the reactive site of BPTI. Chem. Scripta 29A: 27–30

    Google Scholar 

  • Wand A.J. (2001) Dynamic activation of protein function: a view emerging from NMR spectroscopy. Nat. Struct. Biol. 8: 926–931

    Article  Google Scholar 

  • Weigelt J. (1998) Single scan, sensitivity- and gradient-enhanced TROSY for multidimensional NMR experiments. J. Am. Chem. Soc 120: 10778–10779

    Article  Google Scholar 

  • Woessner D.E. (1962) Nuclear spin relaxation in ellipsoids undergoing rotational Brownian motion. J. Chem. Phys. 37: 647–654

    Article  ADS  Google Scholar 

  • Yu L.P., Sun C.H., Song D.Y., Shen J.W., Xu N., Gunasekera A., Hajduk P.J., Olejniczak E.T. (2005) Nuclear magnetic resonance studies of a potassium channel-charybdotoxin complex. Biochemistry 44: 15834–15841

    Article  Google Scholar 

  • Zhou Y., MacKinnon R. (2003) The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates. J. Mol. Biol. 333: 965–975

    Article  Google Scholar 

  • Zhou Y., Morais-Cabral J.H., Kaufman A., MacKinnon R. (2001) Chemistry on ion coordiantion and hydration revealed by a K+ channel-Fab complex at 2.0 Å resolution. Nature 414: 43–48

    Article  ADS  Google Scholar 

  • Zhu G., Xia Y., Nicholson L.K., Sze K.H. (2000) Protein dynamics measurements by TROSY-based NMR experiments. J. Magn. Res. 143: 423–426

    Article  ADS  Google Scholar 

Download references

Acknowledgements

We thank Frank Delaglio for software support, Dennis Torchia (NIDCR/NIH) and Chris Miller (Brandeis University) for helpful discussions and Annie Aniana for assistance in sample preparation. J. H. C. acknowledges the support of a long-term EMBO fellowship. This work was supported by the Intramural Research Program of the NIDDK, NIH, and by the Intramural AIDS-Targeted Antiviral Program of the Office of the Director, NIH.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ad Bax.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s10858-006-9141-7

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chill, J.H., Louis, J.M., Baber, J.L. et al. Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel. J Biomol NMR 36, 123–136 (2006). https://doi.org/10.1007/s10858-006-9071-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-006-9071-4

Keywords

Navigation