Skip to main content
SpringerLink
Log in

Topology and immersion depth of an integral membrane protein by paramagnetic rates from dissolved oxygen

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

Abstract

In studies of membrane proteins, knowledge of protein topology can provide useful insight into both structure and function. In this work, we present a solution NMR method for the measurement the tilt angle and average immersion depth of alpha helices in membrane proteins, from analysis of the paramagnetic relaxation rate enhancements arising from dissolved oxygen. No modification to the micelle or protein is necessary, and the topology of both transmembrane and amphipathic helices are readily determined. We apply this method to the measure the topology of a monomeric mutant of phospholamban (AFA-PLN), a 52-residue membrane protein containing both an amphipathic and a transmembrane alpha helix. In dodecylphosphocholine micelles, the amphipathic helix of AFA-PLN was found to have a tilt angle of 87° ± 1° and an average immersion depth of 13.2 Å. The transmembrane helix was found to have an average immersion depth of 5.4 Å, indicating residues 41 and 42 are closest to the micelle centre. The resolution of paramagnetic relaxation rate enhancements from dissolved oxygen compares favourably to those from Ni (II), a hydrophilic paramagnetic species.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

AFA-PLN:

Phospholamban mutant C36A/C41F/C46A

DOPC:

1,2-dioleoyl-glycero-3-sn-phosphocholine

DOPE:

Dioleylphosphoethanolamine

EPR:

Electron paramagnetic resonance

HSQC:

Heteronuclear single quantum correlation

NOE:

Nuclear overhauser effect

PISEMA:

Polarization inversion spin exchange at magic angle

PLN, wt-PLN:

Phospholamban

PRE:

Paramagnetic relaxation enhancement

RDC:

Residual dipolar coupling

SDS:

Sodium dodecyl sulfate

SERCA:

Sarco(endo)plasmic reticulum calcium ATPase

References

  • Abragam A (1961) The principles of nuclear magnetism. Clarendon Press, Oxford

    Google Scholar 

  • Al-Abdul-Wahid MS, Yu C-H, Batruch I, Evanics F, Pomes R, Prosser RS (2006) A combined NMR and molecular dynamics study of the transmembrane solubility and diffusion rate profile of dioxygen in lipid bilayers. Biochemistry 45:10719–10728

    Article  Google Scholar 

  • Al-Abdul-Wahid MS, Neale C, Pomes R, Prosser RS (2009) A solution NMR approach to the measurement of amphiphile immersion depth and orientation in membrane model systems. J Am Chem Soc 131:6452–6459

    Article  Google Scholar 

  • Al-Abdul-Wahid MS, Evanics F, Prosser RS (2011) Dioxygen transmembrane distributions and partitioning thermodynamics in lipid bilayers and micelles. Biochemistry 50:3975–3983

    Article  Google Scholar 

  • Altenbach C, Marti T, Khorana H, Hubbell W (1990) Transmembrane protein-structure—spin labeling of bacteriorhodopsin mutants. Science 248:1088–1092

    Article  ADS  Google Scholar 

  • Altenbach C, Greenhalgh DA, Khorana HG, Hubbell WL (1994) A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin. Proc Natl Acad Sci USA 91:1667–1671

    Article  ADS  Google Scholar 

  • Arkin I, Rothman M, Ludlam C, Aimoto S, Engelman D, Rothschild K, Smith S (1995) Structural model of the phospholamban ion-channel complex in phospholipid-membranes. J Mol Biol 248:824–834

    Article  Google Scholar 

  • Bax A, Grishaev A (2005) Weak alignment NMR: a hawk-eyed view of biomolecular structure. Curr Opin Struct Biol 15:563–570

    Article  Google Scholar 

  • Bertini I, Gupta YK, Luchinat C, Parigi G, Peana M, Sgheri L, Yuan J (2007) Paramagnetism-based NMR restraints provide maximum allowed probabilities for the different conformations of partially independent protein domains. J Am Chem Soc 129:12786–12794

    Article  Google Scholar 

  • Bokoch MP, Zou Y, Rasmussen SGF, Liu CW, Nygaard R, Rosenbaum DM, Fung JJ, Choi H-J, Thian FS, Kobilka TS, Puglisi JD, Weis WI, Pardo L, Prosser RS, Mueller L, Kobilka BK (2010) Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463:108–112

    Article  ADS  Google Scholar 

  • Boyd J, Hommel U, Campbell ID (1990) Influence of cross-correlation between dipolar and anisotropic chemical-shift relaxation mechanisms upon longitudinal relaxation rates of N-15 in macromolecules. Chem Phys Lett 175:477–482

    Article  ADS  Google Scholar 

  • Buck B, Zamoon J, Kirby TL, DeSilva TM, Karim C, Thomas D, Veglia G (2003) Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies. Protein Expr Purif 30:253–261

    Article  Google Scholar 

  • Chou J, Gaemers S, Howder B, Louis J, Bax A (2001) A simple apparatus for generating stretched polyacrylamide gels, yielding uniform alignment of proteins and detergent micelles. J Biomol NMR 21:377–382

    Article  Google Scholar 

  • Clore GM, Gronenborn AM, Tjandra N (1998) Direct structure refinement against residual dipolar couplings in the presence of rhombicity of unknown magnitude. J Magn Reson 131:159–162

    Article  ADS  Google Scholar 

  • Dalvit C, Hommel U (1995a) Sensitivity-improved detection of protein hydration and its extension to the assignment of fast-exchanging resonances. J Magn Reson B 109:334–338

    Article  Google Scholar 

  • Dalvit C, Hommel U (1995b) New pulsed-field gradient NMR experiments for the detection of bound water in proteins. J Biomol NMR 5:306–310

    Article  Google Scholar 

  • Delaglio F, Grzesiek S, Vuister GW, 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 

  • Evanics F, Hwang PM, Cheng Y, Kay LE, Prosser RS (2006) Topology of an outer-membrane enzyme: measuring oxygen and water contacts in solution NMR studies of PagP. J Am Chem Soc 128:8256–8264

    Article  Google Scholar 

  • Hus JC, Salmon L, Bouvignies G, Lotze J, Blackledge M, Brüschweiler R (2008) 16-Fold degeneracy of peptide plane orientations from residual dipolar couplings: analytical treatment and implications for protein structure determination. J Am Chem Soc 130:15927–15937

    Article  Google Scholar 

  • Huster D, Arnold K, Gawrisch K (1999) Investigation of lipid organization in biological membranes by two-dimensional nuclear overhauser enhancement spectroscopy. J Phys Chem B 103:243–251

    Article  Google Scholar 

  • Jensen MR, Blackledge M (2008) On the origin of NMR dipolar waves in transient helical elements of partially folded proteins. J Am Chem Soc 130:11266–11267

    Article  Google Scholar 

  • Johnson BA, Blevins RA (1994) NMR view—a computer-program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614

    Article  Google Scholar 

  • Karim CB, Marquardt CG, Stamm JD, Barany G, Thomas DD (2000) Synthetic null-cysteine phospholamban analogue and the corresponding transmembrane domain inhibit the Ca-ATPase. Biochemistry 39:10892–10897

    Article  Google Scholar 

  • Kay LE, Nicholson LK, Delaglio F, Bax A, Torchia DA (1992) Pulse sequences for removal of the effects of cross-correlation between dipolar and chemical-shift anisotropy relaxation mechanism on the measurement of heteronuclear T1 and T2 values in proteins. J Magn Reson 97:359–375

    Article  Google Scholar 

  • Kimura Y, Asahi M, Kurzydlowski K, Tada M, MacLennan D (1998) Phospholamban domain Ib mutations influence functional interactions with the Ca2+-ATPase isoform of cardiac sarcoplasmic reticulum. J Biol Chem 273:14238–14241

    Article  Google Scholar 

  • Kitevski-Leblanc JL, Evanics F, Prosser RS (2009) Approaches for the measurement of solvent exposure in proteins by F-19 NMR. J Biomol NMR 45:255–264

    Article  Google Scholar 

  • Losonczi J, Olejniczak E, Betz S, Harlan J, Mack J, Fesik S (2000) NMR studies of the anti-apoptotic protein Bcl-x(L) in micelles. Biochemistry 39:11024–11033

    Article  Google Scholar 

  • Maclennan DH, Kranias EG (2003) Calcium: phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol 4:566–577

    Article  Google Scholar 

  • Madl T, Bermel W, Zangger K (2009) Use of relaxation enhancements in a paramagnetic environment for the structure determination of proteins using NMR spectroscopy. Angew Chem Int Ed 48:8259–8262

    Article  Google Scholar 

  • Marrink S, Berendsen H (1994) Simulation of water transport through a lipid-membrane. J Phys Chem Us 98:4155–4168

    Article  Google Scholar 

  • Marrink S, Berendsen H (1996) Permeation process of small molecules across lipid membranes studied by molecular dynamics simulations. J Phys Chem Us 100:16729–16738

    Article  Google Scholar 

  • Marsh D (2001) Polarity and permeation profiles in lipid membranes. Proc Natl Acad Sci USA 98:7777–7782

    Article  ADS  Google Scholar 

  • Pauling L, Corey RB, Branson HR (1951) The structure of proteins—2 hydrogen-bonded helical configurations of the polypeptide chain. Proc Natl Acad Sci USA 37:205–211

    Article  ADS  Google Scholar 

  • Pintacuda G, Otting G (2002) Identification of protein surfaces by NMR measurements with a paramagnetic Gd(III) chelate. J Am Chem Soc 124:372–373

    Article  Google Scholar 

  • Prosser RS, Luchette PA, Westerman PW (2000) Using O2 to probe membrane immersion depth by 19F NMR. Proc Natl Acad Sci USA 97:9967–9971

    Article  ADS  Google Scholar 

  • Respondek M, Madl T, Goebl C, Golser R, Zangger K (2007) Mapping the orientation of helices in micelle-bound peptides by paramagnetic relaxation waves. J Am Chem Soc 129:5228–5234

    Article  Google Scholar 

  • Rosen M, Gardner K, Willis R, Parris W, Pawson T, Kay L (1996) Selective methyl group protonation of perdeuterated proteins. J Mol Biol 263:627–636

    Article  Google Scholar 

  • Shi L, Traaseth NJ, Verardi R, Gustavsson M, Gao J, Veglia G (2011) Paramagnetic-based NMR restraints lift residual dipolar coupling degeneracy in multidomain detergent-solubilized membrane proteins. J Am Chem Soc 133:2232–2241

    Article  Google Scholar 

  • Simmerman H, Kobayashi Y, Autry J, Jones L (1996) A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure. J Biol Chem 271:5941–5946

    Article  Google Scholar 

  • Smith S, Kawakami T, Liu W, Ziliox M, AIMOTO S (2001) Helical structure of phospholamban in membrane bilayers. J Mol Biol 313:1139–1148

    Article  Google Scholar 

  • Steinhoff H, Mollaaghababa R, Altenbach C, Hideg K, Krebs M, Khorana H, Hubbell W (1994) Time-resolved detection of structural-changes during the photocycle of spin-labeled bacteriorhodopsin. Science 266:105–107

    Article  ADS  Google Scholar 

  • Steinhoff H, Mollaaghababa R, Altenbach C, Khorana H, Hubbell W (1995) Site-directed spin-labeling studies of structure and dynamics in bacteriorhodopsin. Biophys Chem 56:89–94

    Article  Google Scholar 

  • Su Y, Mani R, Hong M (2008) Asymmetric insertion of membrane proteins in lipid bilayers by solid-state NMR paramagnetic relaxation enhancement: a cell-penetrating peptide example. J Am Chem Soc 130:8856–8864

    Article  Google Scholar 

  • Teng C, Hinderliter B, Bryant R (2006) Oxygen accessibility to ribonuclease A: quantitative interpretation of nuclear spin relaxation induced by a freely diffusing paramagnet. J Phys Chem A 110:580–588

    Article  Google Scholar 

  • Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114

    Article  ADS  Google Scholar 

  • Tjandra N, Marquardt J, Clore GM (2000) Direct refinement against proton-proton dipolar couplings in NMR Structure determination of macromolecules. J Magn Reson 142:393–396

    Article  ADS  Google Scholar 

  • Traaseth NJ, Buffy JJ, Zamoon J, Veglia G (2006) Structural dynamics and topology of phospholamban in oriented lipid bilayers using multidimensional solid-state NMR. Biochemistry 45:13827–13834

    Article  Google Scholar 

  • Traaseth NJ, Verardi R, Torgersen KD, Karim CB, Thomas DD, Veglia G (2007) Spectroscopic validation of the pentameric structure of phospholamban. Proc Natl Acad Sci USA 104:14676–14681

    Article  ADS  Google Scholar 

  • Traaseth NJ, Shi L, Verardi R, Mullen DG, Barany G, Veglia G (2009) Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach. Proc Natl Acad Sci 106:10165

    Article  ADS  Google Scholar 

  • Verardi R, Shi L, Traaseth NJ, Walsh N, Veglia G (2011) Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method. Proc Natl Acad Sci USA 108:9101–9106

    Article  ADS  Google Scholar 

  • Weiss R (1970) Solubility of nitrogen, oxygen and argon in water and seawater. Deep Sea Res 17:721–735

    Google Scholar 

  • Wu CH, Ramamoorthy A, Opella SJ (1994) High-resolution heteronuclear dipolar solid-state NMR-spectroscopy. J Magn Reson Ser A 109:270–272

    Article  Google Scholar 

  • Zamoon J, Mascioni A, Thomas DD, Veglia G (2008) NMR solution structure and topological orientation of monomeric phospholamban in dodecylphosphocholine micelles. Biophys J 85:2589–2598

    Article  Google Scholar 

  • Zangger K, Respondek M, Goebl C, Hohlweg W, Rasmussen K, Grampp G, Madl T (2009) Positioning of micelle-bound peptides by paramagnetic relaxation enhancements. J Phys Chem B 113:4400–4406

    Article  Google Scholar 

Download references

Acknowledgments

RSP acknowledges NSERC, and the Ontario government for financial support through the NSERC discovery and Provincial Research Excellence Award (PREA) programs. This work was in part supported by the National Institute of Health (GM64742) to GV.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Toronto Mississauga, Mississauga, ON, L5L 1C6, Canada

    M. Sameer Al-Abdul-Wahid & R. Scott Prosser

  2. Department of Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA

    Raffaello Verardi & Gianluigi Veglia

  3. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA

    Raffaello Verardi & Gianluigi Veglia

  4. Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada

    R. Scott Prosser

Authors
  1. M. Sameer Al-Abdul-Wahid
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Raffaello Verardi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gianluigi Veglia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. Scott Prosser
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. Scott Prosser.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Al-Abdul-Wahid, M.S., Verardi, R., Veglia, G. et al. Topology and immersion depth of an integral membrane protein by paramagnetic rates from dissolved oxygen. J Biomol NMR 51, 173 (2011). https://doi.org/10.1007/s10858-011-9551-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s10858-011-9551-z

Keywords

Search

Navigation