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Solid-State NMR Approaches to Study Protein Structure and Protein–Lipid Interactions

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Lipid-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2003))

Abstract

Solid-state NMR spectroscopy has been developed for the investigation of membrane-associated polypeptides and remains one of the few techniques to reveal high-resolution structural information in liquid-disordered phospholipid bilayers. In particular, oriented samples have been used to investigate the structure, dynamics and topology of membrane polypeptides. Much of the previous solid-state NMR work has been developed and performed on peptides but the technique is constantly expanding towards larger membrane proteins. Here, a number of protocols are presented describing among other the reconstitution of membrane proteins into oriented membranes, monitoring membrane alignment by 31P solid-state NMR spectroscopy, investigations of the protein by one- and two-dimensional 15N solid-state NMR and measurements of the lipid order parameters using 2H solid-state NMR spectroscopy. Using such methods solid-state NMR spectroscopy has revealed a detailed picture of the ensemble of both lipids and proteins and their mutual interdependence in the bilayer environment.

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References

  1. Wallin E, von Heijne G (1998) Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci 7:1029–1038

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Boyd D, Schierle C, Beckwith J (1998) How many membrane proteins are there. Protein Sci 7:201–205

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Xiang J, Chun E et al (2016) Successful strategies to determine high-resolution structures of GPCRs. Trends Pharmacol Sci 37:1055–1069

    CAS  PubMed  Google Scholar 

  4. Latorraca NR, Venkatakrishnan AJ, Dror RO (2017) GPCR dynamics: structures in motion. Chem Rev 117:139–155

    CAS  PubMed  Google Scholar 

  5. Vostrikov VV, Soller KJ et al (2015) Effects of naturally occurring arginine 14 deletion on phospholamban conformational dynamics and membrane interactions. Biochim Biophys Acta 1848:315–322

    CAS  PubMed  Google Scholar 

  6. Michalek M, Salnikov E et al (2013) Structure and topology of the huntingtin 1–17 membrane anchor by a combined solution and solid-state NMR approach. Biophys J 105:699–710

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Gopinath T, Mote KR, Veglia G (2015) Simultaneous acquisition of 2D and 3D solid-state NMR experiments for sequential assignment of oriented membrane protein samples. J Biomol NMR 62(1):53–61

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Baker LA, Baldus M (2014) Characterization of membrane protein function by solid-state NMR spectroscopy. Curr Opin Struct Biol 27:48–55

    CAS  PubMed  Google Scholar 

  9. Das N, Dai J et al (2015) Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis, in lipid bilayers. Proc Natl Acad Sci U S A 112:E119–E126

    CAS  PubMed  Google Scholar 

  10. Saurel O, Iordanov I et al (2017) Local and global dynamics in Klebsiella pneumoniae outer membrane protein a in lipid bilayers probed at atomic resolution. J Am Chem Soc 139:1590–1597

    CAS  PubMed  Google Scholar 

  11. Lehnert E, Mao J et al (2016) Antigenic peptide recognition on the human ABC transporter TAP resolved by DNP-enhanced solid-state NMR spectroscopy. J Am Chem Soc 138(42):13967–13974

    CAS  PubMed  Google Scholar 

  12. Naito A, Matsumori N, Ramamoorthy A (2018) Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy. Biochim Biophys Acta 1862:307–323

    CAS  Google Scholar 

  13. Bechinger B, Kinder R et al (1999) Peptide structural analysis by solid-state NMR spectroscopy. Biopolymers 51:174–190

    CAS  PubMed  Google Scholar 

  14. Aisenbrey C, Bertani P, Bechinger B (2010) Solid-state NMR investigations of membrane-associated antimicrobial peptides. In: Guiliani A, Rinaldi AC (eds) Antimicrobial peptides. Humana Press, Springer, New York, pp 209–233

    Google Scholar 

  15. Bechinger B, Sizun C (2003) Alignment and structural analysis of membrane polypeptides by 15N and 31P solid-state NMR spectroscopy. Concepts Magn Reson 18A:130–145

    CAS  Google Scholar 

  16. Salnikov E, Aisenbrey C et al (2010) Solid-state NMR approaches to measure topological equilibria and dynamics of membrane polypeptides. Biochim Biophys Acta 1798:258–265

    CAS  PubMed  Google Scholar 

  17. Bechinger B, Resende JM, Aisenbrey C (2011) The structural and topological analysis of membrane-associated polypeptides by oriented solid-state NMR spectroscopy: established concepts and novel developments. Biophys Chem 153:115–125

    CAS  PubMed  Google Scholar 

  18. Aisenbrey C, Bechinger B (2004) Tilt and rotational pitch angles of membrane-inserted polypeptides from combined 15N and 2H solid-state NMR spectroscopy. Biochemistry 43:10502–10512

    CAS  PubMed  Google Scholar 

  19. Cullis PR, De Kruijff B (1979) Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim Biophys Acta 559:399–420

    CAS  PubMed  Google Scholar 

  20. Kim C, Spano J et al (2009) Evidence of pores and thinned lipid bilayers induced in oriented lipid membranes interacting with the antimicrobial peptides, magainin-2 and aurein-3.3. Biochim Biophys Acta 1788:1482–1496

    CAS  PubMed  Google Scholar 

  21. Bechinger B, Salnikov ES (2012) The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy. Chem Phys Lipids 165:282–301

    CAS  PubMed  Google Scholar 

  22. Salnikov ES, Mason AJ, Bechinger B (2009) Membrane order perturbation in the presence of antimicrobial peptides by 2H solid-state NMR spectroscopy. Biochimie 91:743

    Google Scholar 

  23. Harmouche N, Pachler M et al (2018) Lipid-mediated interactions between the amphipathic antimicrobial peptides magainin 2 and PGLa in phospholipid bilayers. Biophys J 115(6):1033–1044

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Mason AJ, Martinez A et al (2006) The antibiotic and DNA-transfecting peptide LAH4 selectively associates with, and disorders, anionic lipids in mixed membranes. FASEB J 20:320–322

    CAS  PubMed  Google Scholar 

  25. Seelig J, Macdonald PM, Scherer PG (1987) Phospholipid head groups as sensors of electric charge in membranes. Biochemistry 26:7535–7541

    CAS  PubMed  Google Scholar 

  26. Bechinger B, Seelig J (1991) Interaction of electric dipoles with phospholipid head groups. A 2H and 31P NMR study of phloretin and phloretin analogues in phosphatidylcholine membranes. Biochemistry 30:3923–3929

    CAS  PubMed  Google Scholar 

  27. Wolf J, Aisenbrey C et al (2017) pH-dependent membrane interactions of the histidine-rich cell penetrating peptide LAH4-L1. Biophys J 113(6):1290–1300

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Ravula T, Hardin NZ et al (2017) pH resistant monodispersed polymer-lipid nanodiscs. Angew Chem Int Ed Engl 57(5):1342–1345

    Google Scholar 

  29. Loudet C, Diller A et al (2010) Biphenyl phosphatidylcholine: a promoter of liposome deformation and bicelle collective orientation by magnetic fields. Prog Lipid Res 49:289–297

    CAS  PubMed  Google Scholar 

  30. Aisenbrey C, Sudheendra US et al (2007) Helix orientations in membrane-associated Bcl-XL determined by 15N solid-state NMR spectroscopy. Eur Biophys J 37:71–80

    CAS  PubMed  Google Scholar 

  31. Nedelkina S, Gokce I et al (2008) High-yield expression and purification of soluble forms of the anti-apoptotic Bcl-x(L) and Bcl-2 as TolAIII-fusion proteins. Protein Expr Purif 29:1633–1644

    Google Scholar 

  32. Aisenbrey C, Cusan M et al (2008) Specific isotope labeling of colicin E1 and B channel domains for membrane topological analysis by oriented solid-state NMR spectroscopy. Chembiochem 9:944–951

    CAS  PubMed  Google Scholar 

  33. Su Y, Andreas L, Griffin RG (2015) Magic angle spinning NMR of proteins: high-frequency dynamic nuclear polarization and H detection. Annu Rev Biochem 84:465–497

    CAS  PubMed  Google Scholar 

  34. Yamamoto K, Caporini MA et al (2015) Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization. Biochim Biophys Acta Biomembranes 1848:342–349

    CAS  Google Scholar 

  35. Kaplan M, Pinto C et al (2016) Nuclear magnetic resonance (NMR) applied to membrane-protein complexes. Q Rev Biophys 49:e15

    PubMed  Google Scholar 

  36. Salnikov E, Sarrouj H et al (2015) Solid-state NMR/Dynamic Nuclear Polarization of planar supported lipid bilayers. J Phys Chem B 119:14574–14583

    CAS  PubMed  Google Scholar 

  37. Salnikov ES, Abel S et al (2017) Dynamic nuclear polarization/solid-state NMR spectroscopy of membrane polypeptides: free-radical optimization for matrix-free lipid bilayer samples. ChemPhysChem 18:2103–2113

    CAS  PubMed  Google Scholar 

  38. Salnikov E, Rosay M et al (2010) Solid-state NMR spectroscopy of oriented membrane polypeptides at 100 K with signal enhancement by dynamic nuclear polarization. J Am Chem Soc 132:5940–5941

    CAS  PubMed  Google Scholar 

  39. Bechinger B, Opella SJ (1991) Flat-coil probe for NMR spectroscopy of oriented membrane samples. J Magn Reson 95:585–588

    Google Scholar 

  40. Nielsen NC, Daugaard P et al (1995) A flat-coil NMR probe with hydration control of oriented phospholipid-bilayer samples. J Biomol NMR 5:311–314

    CAS  PubMed  Google Scholar 

  41. Salnikov ES, Aisenbrey C et al (2016) Membrane topologies of the PGLa antimicrobial peptide and a transmembrane anchor sequence by Dynamic Nuclear Polarization/solid-state NMR spectroscopy. Sci Rep 6:20895

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Rosay M, Tometich L et al (2010) Solid-state dynamic nuclear polarization at 263 GHz: spectrometer design and experimental results. Phys Chem Chem Phys 12:5850–5860

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Bechinger B (2011) Insights into the mechanisms of action of host defence peptides from biophysical and structural investigations. J Pept Sci 17:306–314

    CAS  PubMed  Google Scholar 

  44. Bechinger B, Bertani P et al (2010) The structural and topological analysis of membrane polypeptides by oriented solid-state NMR spectroscopy: sample preparation and theory. In: Miguel C (ed) Membrane-active peptides: methods and results on structure and function. International University Line, La Jolla, California, pp 196–215

    Google Scholar 

  45. Chenal A, Prongidi-Fix L et al (2009) Deciphering membrane insertion of the diphtheria toxin T domain by specular neutron reflectometry and solid-state NMR spectroscopy. J Mol Biol 391:872–883

    CAS  PubMed  Google Scholar 

  46. Lakey JH, Slatin SL (2001) Pore-forming colicins and their relatives. Curr Top Microbiol Immunol 257:131–161

    CAS  PubMed  Google Scholar 

  47. Raja M, Vales E (2011) Improved technique for reconstituting incredibly high and soluble amounts of tetrameric K(+) channel in natural membranes. J Membr Biol 241:141–144

    CAS  PubMed  Google Scholar 

  48. Highsmith S (1990) On the mechanism of detergent modification of myosin structure and function. J Biochem 107:554–558

    CAS  PubMed  Google Scholar 

  49. Sanders CR, Prosser RS (1998) Bicelles - a model membrane system for all seasons. Structure 6:1227–1234

    CAS  PubMed  Google Scholar 

  50. Salnikov ES, Anantharamaiah GM, Bechinger B (2018) Supramolecular organization of apolipoprotein A-I - derived peptides within disc-like arrangements. Biophys J 115(3):467–477

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Berger S, Braun S (2004) 200 and more basic NMR experiments: a practical course. Wiley-VCH, Weinheim

    Google Scholar 

  52. Rance M, Byrd RA (1983) Obtaining high-fidelity spin-1/2 powder spectra in anisotropic media: phase-cycled Hahn echo spectroscopy. J Magn Reson 52:221–240

    CAS  Google Scholar 

  53. Bertani P, Raya J, Bechinger B (2014) 15N chemical shift referencing in solid state NMR. Solid State Nucl Magn Reson 61–62:15–18

    PubMed  Google Scholar 

  54. Ramamoorthy A, Wei Y, Lee D (2004) PISEMA solid-state NMR spectroscopy. Annu Rep NMR Spectrosc 52:1–52

    Google Scholar 

  55. Loffredo MR, Ghosh A et al (2017) Membrane perturbing activities and structural properties of the frog-skin derived peptide Esculentin-1a(1-21)NH2 and its Diastereomer Esc(1-21)-1c: correlation with their antipseudomonal and cytotoxic activity. Biochim Biophys Acta 1859:2327–2339

    CAS  Google Scholar 

  56. Davis JH, Jeffrey KR et al (1976) Quadrupolar echo deuteron magnetic resonance spectroscopy in ordered hydrocarbon chains. Chem Phys Lett 42:390–394

    CAS  Google Scholar 

  57. Salnikov E, Bechinger B (2011) Lipid-controlled peptide topology and interactions in bilayers: structural insights into the synergistic enhancement of the antimicrobial activities of PGLa and magainin 2. Biophys J 100:1473–1480

    CAS  PubMed  PubMed Central  Google Scholar 

  58. O’Brian FEM (1948) The control of humidity by saturated salt solutions. J Sci Instr 25:73–76

    Google Scholar 

  59. Liao SY, Lee M et al (2016) Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location. J Biomol NMR 64:223–237

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Becker-Baldus J, Bamann C et al (2015) Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy. Proc Natl Acad Sci U S A 112:9896–9901

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Salnikov ES, Ouari O et al (2012) Developing DNP/solid-state NMR spectroscopy of oriented membranes. Appl Magn Reson 43:91–106

    CAS  Google Scholar 

  62. Ottiger M, Bax A (1998) Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules. J Biomol NMR 12:361–372

    CAS  PubMed  Google Scholar 

  63. Salnikov ES, Friedrich H et al (2009) Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy. Biophys J 96:86–100

    CAS  PubMed  Google Scholar 

  64. Traikia M, Warschawski DE et al (2000) Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and P-31-nuclear magnetic resonance. Eur Biophys J Biophys 29:184–195

    CAS  Google Scholar 

  65. Yokogawa M, Takeuchi K, Shimada I (2005) Bead-linked proteoliposomes: a reconstitution method for NMR analyses of membrane protein-ligand interactions. J Am Chem Soc 127:12021–12027

    CAS  PubMed  Google Scholar 

  66. Gopinath T, Mote KR, Veglia G (2013) Sensitivity and resolution enhancement of oriented solid-state NMR: application to membrane proteins. Prog Nucl Magn Reson Spectrosc 75:50–68

    CAS  PubMed  Google Scholar 

  67. Jayanthi S, Sinha N, Ramanathan KV (2010) 2(4)-SEMA as a sensitive and offset compensated SLF sequence. J Magn Reson 207:206–212

    CAS  PubMed  Google Scholar 

  68. Hjelmeland LM, Nebert DW, Osborne JC (1983) Sulfobetaine derivatives of bile-acids - non-denaturing surfactants for membrane biochemistry. Anal Biochem 130:72–82

    CAS  PubMed  Google Scholar 

  69. Womack MD, Kendall DA, Macdonald RC (1983) Detergent effects on enzyme-activity and solubilization of lipid bilayer-membranes. Biochim Biophys Acta 733:210–215

    CAS  PubMed  Google Scholar 

  70. Banerjee P, Joo JB et al (1995) Differential solubilization of lipids along with membrane-proteins by different classes of detergents. Chem Phys Lipids 77:65–78

    CAS  PubMed  Google Scholar 

  71. Hjelmeland LM (1980) A non-denaturing zwitterionic detergent for membrane biochemistry - design and synthesis. Proc Natl Acad Sci U S A 77:6368–6370

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Vanaken T, Foxallvanaken S et al (1986) Alkyl glycoside detergents - synthesis and applications to the study of membrane-proteins. Methods Enzymol 125:27–35

    CAS  PubMed  Google Scholar 

  73. Jacobs JJ, Anderson RA, Watson TR (1971) Interactions in phenol-sodium dodecyl sulphate-water systems. J Pharm Pharmacol 23:148–149

    CAS  PubMed  Google Scholar 

  74. Lauterwein J, Bosch C et al (1979) Physicochemical studies of the protein-lipid interactions in melittin-containing micelles. Biochim Biophys Acta 556:244–264

    CAS  PubMed  Google Scholar 

  75. Palladino P, Rossi F, Ragone R (2010) Effective critical micellar concentration of a zwitterionic detergent: a fluorimetric study on n-Dodecyl phosphocholine. J Fluoresc 20:191–196

    CAS  PubMed  Google Scholar 

  76. Ross J, Olivier JP (1959) A new method for the determination of critical micelle concentrations of uncharged association colloids in aqueous or in non-aqueous solution. J Phys Chem 63:1671–1674

    CAS  Google Scholar 

  77. Dixon AM, Venable RM et al (2002) Micelle-bound conformation of a hairpin-forming peptide: combined NMR and molecular dynamics study. Biopolymers 65:284–298

    CAS  PubMed  Google Scholar 

  78. Lorber B, Bishop JB, Delucas LJ (1990) Purification of octyl beta-D-glucopyranoside and reestimation of its micellar size. Biochim Biophys Acta 1023:254–265

    CAS  PubMed  Google Scholar 

  79. Hierrezuelo JM, Aguiar J, Ruiz CC (2005) Micellar properties of a mixed surfactant system constituted by n-octyl-beta-D-thioglucopyranoside and sodium dodecyl sulphate. Colloid Surf A 264:29–36

    CAS  Google Scholar 

  80. Molina-Bolivar JA, Hierrezuelo JM, Ruiz CC (2006) Effect of NaCl on the self-aggregation of n-octyl beta-D-thioglucopyranoside in aqueous medium. J Phys Chem B 110:12089–12095

    CAS  PubMed  Google Scholar 

  81. Coll H (1970) Study of ionic surfactants by membrane osmometry. J Phys Chem 74:520–528

    CAS  Google Scholar 

  82. le Maire M, Champeil P, Moller JV (2000) Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508:86–111

    PubMed  Google Scholar 

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Acknowledgments

The help and comments by Philippe Bertani on the nutation and PISEMA experiments are gratefully acknowledged. B.B. is most grateful to Gianluigi Veglia and Gopinath Tata from the University of Minnesota NMR center for the in-depth discussions and exchange during his sabbatical stay at the University of Minnesota School of Dentistry that was generously supported by School’s Lasby Visiting Professor Fellowship and the University of Strasbourg. We are grateful for the financial support by the Deutsche Forschungsgemeinschaft (postdoctoral grant to M.M.), the Agence Nationale de la Recherche (projects membraneDNP 12-BSV5-0012, MemPepSyn 14-CE34-0001-01, InMembrane 15-CE11-0017-01, Biosupramol 17-CE18-0033-3 and the LabEx Chemistry of Complex Systems 10-LABX-0026_CSC), the University of Strasbourg, the CNRS, the Région Alsace and the RTRA International Center of Frontier Research in Chemistry, the IRTG Soft Matter Science, the French Foundation for Medical Research (FRM), and the American Foundation for Research on Huntington’s Disease (CHDI). B.B. is grateful to the Institut Universitaire de France for providing additional time to be dedicated to research.

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Authors and Affiliations

  1. Institut de Chimie, CNRS, Université de Strasbourg, UMR 7177, Strasbourg, France

    Christopher Aisenbrey, Evgeniy S. Salnikov, Jesus Raya, Matthias Michalek & Burkhard Bechinger

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  1. Christopher Aisenbrey
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  2. Evgeniy S. Salnikov
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  3. Jesus Raya
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  4. Matthias Michalek
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  5. Burkhard Bechinger
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Correspondence to Burkhard Bechinger .

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  1. Department of Biophysics, Institute of Biology, FB10 and Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Kassel, Germany

    Jörg H. Kleinschmidt

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Aisenbrey, C., Salnikov, E.S., Raya, J., Michalek, M., Bechinger, B. (2019). Solid-State NMR Approaches to Study Protein Structure and Protein–Lipid Interactions. In: Kleinschmidt, J. (eds) Lipid-Protein Interactions. Methods in Molecular Biology, vol 2003. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9512-7_23

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  • DOI: https://doi.org/10.1007/978-1-4939-9512-7_23

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