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Single molecule techniques for the study of membrane proteins

Abstract

Single molecule techniques promise novel information about the properties and behavior of individual particles, thus enabling access to molecular heterogeneities in biological systems. Their recent developments to accommodate membrane studies have significantly deepened the understanding of membrane proteins. In this short review, we will describe the basics of the three most common single-molecule techniques used on membrane proteins: fluorescence correlation spectroscopy, single particle tracking, and atomic force microscopy. We will discuss the most relevant findings made during the recent years and their contribution to the membrane protein field.

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References

  1. Bacia K, Majoul IV, Schwille P (2002) Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. Biophys J 83:1184–1193

  2. Bacia K, Scherfeld D, Kahya N, Schwille P (2004a) Fluorescence correlation spectroscopy relates rafts in model and native membranes. Biophys J 87:1034–1043

  3. Bacia K, Schuette CG, Kahya N, Jahn R, Schwille P (2004b) SNAREs prefer liquid-disordered over “raft” (liquid-ordered) domains when reconstituted into giant unilamellar vesicles. J Biol Chem 279:37951–37955

  4. Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3:83–89

  5. Benda A, Benes M, Marecek V, Lhotsky A, Hermens WT, Hof M (2003) How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. Langmuir 19:4120–4126

  6. Burns AR, Frankel DJ, Buranda T (2005) Local mobility in lipid domains of supported bilayers characterized by atomic force microscopy and fluorescence correlation spectroscopy. Biophys J 89:1081–1093

  7. Buzhynskyy N, Hite RK, Walz T, Scheuring S (2007) The supramolecular architecture of junctional microdomains in native lens membranes. EMBO Rep 8:51–55

  8. Chiantia S, Kahya N, Ries J, Schwille P (2006a) Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS. Biophys J 90:4500–4508

  9. Chiantia S, Ries J, Kahya N, Schwille P (2006b) Combined AFM and two-focus SFCS study of Raft-exhibiting model membranes. ChemPhysChem 7:2409–2418

  10. Cisneros DA, Oesterhelt D, Muller DJ (2005) Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin. Structure 13:235–242

  11. Doeven MK, Folgering JH, Krasnikov V, Geertsma ER, van den BG, Poolman B (2005) Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles. Biophys J 88:1134–1142

  12. Douglass AD, Vale RD (2005) Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121:937–950

  13. Fotiadis D, Suda K, Tittmann P, Jeno P, Philippsen A, Muller DJ, Gross H, Engel A (2002) Identification and structure of a putative Ca2+-binding domain at the C terminus of AQP1. J Mol Biol 318:1381–1394

  14. Gakamsky DM, Luescher IF, Pramanik A, Kopito RB, Lemonnier F, Vogel H, Rigler R, Pecht I (2005) CD8 kinetically promotes ligand binding to the T-cell antigen receptor. Biophys J 89:2121–2133

  15. Girard P, Pecreaux J, Lenoir G, Falson P, Rigaud JL, Bassereau P (2004) A new method for the reconstitution of membrane proteins into giant unilamellar vesicles. Biophys J 87:419–429

  16. Gonçalves RP, Agnus G, Sens P, Houssin C, Bartenlian B, Scheuring S (2006) Two-chamber AFM: probing membrane proteins separating two aqueous compartments. Nat Methods 3:1007–1012

  17. Haustein E, Schwille P (2004) Single-molecule spectroscopic methods. Curr Opin Struct Biol 14:531–540

  18. Hegener O, Jordan R, Haberlein H (2004) Dye-labeled benzodiazepines: development of small ligands for receptor binding studies using fluorescence correlation spectroscopy. J Med Chem 47:3600–3605

  19. Humpolickova J, Gielen E, Benda A, Fagulova V, Vercammen J, Vandeven M, Hof M, Ameloot M, Engelborghs Y (2006) Probing diffusion laws within cellular membranes by Z-scan fluorescence correlation spectroscopy. Biophys J 91:L23–L25

  20. Jacquier V, Prummer M, Segura JM, Pick H, Vogel H (2006) Visualizing odorant receptor trafficking in living cells down to the single-molecule level. Proc Natl Acad Sci USA 103:14325–14330

  21. Janovjak H, Struckmeier J, Hubain M, Kedrov A, Kessler M, Muller DJ (2004) Probing the energy landscape of the membrane protein bacteriorhodopsin. Structure 12:871–879

  22. Janovjak H, Kedrov A, Cisneros DA, Sapra KT, Struckmeier J, Muller DJ (2006) Imaging and detecting molecular interactions of single transmembrane proteins. Neurobiol Aging 27:546–561

  23. Johnson CK, Osborn KD, Allen MW, Slaughter BD (2005) Single-molecule fluorescence spectroscopy: new probes of protein function and dynamics. Physiology (Bethesda) 20:10–14

  24. Kahya N (2006) Targeting membrane proteins to liquid-ordered phases: molecular self-organization explored by fluorescence correlation spectroscopy. Chem Phys Lipids 141:158–168

  25. Kahya N, Wiersma DA, Poolman B, Hoekstra D (2002) Spatial organization of bacteriorhodopsin in model membranes. Light-induced mobility changes. J Biol Chem 277:39304–39311

  26. Kahya N, Brown DA, Schwille P (2005) Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles. Biochemistry 44:7479–7489

  27. Kalvodova L, Kahya N, Schwille P, Ehehalt R, Verkade P, Drechsel D, Simons K (2005) Lipids as modulators of proteolytic activity of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro. J Biol Chem 280:36815–36823

  28. Kedrov A, Ziegler C, Janovjak H, Kuhlbrandt W, Muller DJ (2004) Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy. J Mol Biol 340:1143–1152

  29. Kedrov A, Krieg M, Ziegler C, Kuhlbrandt W, Muller DJ (2005) Locating ligand binding and activation of a single antiporter. EMBO Rep 6:668–674

  30. Kedrov A, Janovjak H, Ziegler C, Kuhlbrandt W, Muller DJ (2006a) Observing folding pathways and kinetics of a single sodium-proton antiporter from Escherichia coli. J Mol Biol 355:2–8

  31. Kedrov A, Ziegler C, Muller DJ (2006b) Differentiating ligand and inhibitor interactions of a single antiporter. J Mol Biol 362:925–932

  32. Kessler M, Gaub HE (2006) Unfolding barriers in bacteriorhodopsin probed from the cytoplasmic and the extracellular side by AFM. Structure 14:521–527

  33. Kessler M, Gottschalk KE, Janovjak H, Muller DJ, Gaub HE (2006) Bacteriorhodopsin folds into the membrane against an external force. J Mol Biol 357:644–654

  34. Kienberger F, Ebner A, Gruber HJ, Hinterdorfer P (2006) Molecular recognition imaging and force spectroscopy of single biomolecules. Acc Chem Res 39:29–36

  35. Kusumi A, Ike H, Nakada C, Murase K, Fujiwara T (2005a) Single-molecule tracking of membrane molecules: plasma membrane compartmentalization and dynamic assembly of raft-philic signaling molecules. Semin Immunol 17:3–21

  36. Kusumi A, Nakada C, Ritchie K, Murase K, Suzuki K, Murakoshi H, Kasai RS, Kondo J, Fujiwara T (2005b) Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. Annu Rev Biophys Biomol Struct 34:351–378

  37. Larson DR, Ma YM, Vogt VM, Webb WW (2003) Direct measurement of Gag-Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy. J Cell Biol 162:1233–1244

  38. Lenne PF, Wawrezinieck L, Conchonaud F, Wurtz O, Boned A, Guo XJ, Rigneault H, He HT, Marguet D (2006) Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork. EMBO J 25:3245–3256

  39. Lieto AM, Cush RC, Thompson NL (2003) Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy. Biophys J 85:3294–3302

  40. Lill Y, Martinez KL, Lill MA, Meyer BH, Vogel H, Hecht B (2005) Kinetics of the initial steps of g protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging. ChemPhysChem 6:1633–1640

  41. Lin CW, Yan F, Shimamura S, Barg S, Shyng SL (2005) Membrane phosphoinositides control insulin secretion through their effects on ATP-sensitive K+ channel activity. Diabetes 54:2852–2858

  42. Liu F, Arce FT, Ramachandran S, Lal R (2006) Nanomechanics of hemichannel conformations: connexin flexibility underlying channel opening and closing. J Biol Chem 281:23207–23217

  43. Lommerse PH, Snaar-Jagalska BE, Spaink HP, Schmidt T (2005) Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation. J Cell Sci 118:1799–1809

  44. Meissner O, Haberlein H (2003) Lateral mobility and specific binding to GABA(A) receptors on hippocampal neurons monitored by fluorescence correlation spectroscopy. Biochemistry 42:1667–1672

  45. Moller C, Fotiadis D, Suda K, Engel A, Kessler M, Muller DJ (2003) Determining molecular forces that stabilize human aquaporin-1. J Struct Biol 142:369–378

  46. Morris VJ, Kirby AR, Gunning AP (1999) Atomic force microscopy for biologists. Imperial College Press, London

  47. Muller DJ, Hand GM, Engel A, Sosinsky GE (2002a) Conformational changes in surface structures of isolated connexin 26 gap junctions. EMBO J 21:3598–3607

  48. Muller DJ, Kessler M, Oesterhelt F, Moller C, Oesterhelt D, Gaub H (2002b) Stability of bacteriorhodopsin alpha-helices and loops analyzed by single-molecule force spectroscopy. Biophys J 83:3578–3588

  49. Muller DJ, Sapra KT, Scheuring S, Kedrov A, Frederix PL, Fotiadis D, Engel A (2006) Single-molecule studies of membrane proteins. Curr Opin Struct Biol 16:489–495

  50. Murakoshi H, Iino R, Kobayashi T, Fujiwara T, Ohshima C, Yoshimura A, Kusumi A (2004) Single-molecule imaging analysis of Ras activation in living cells. Proc Natl Acad Sci USA 101:7317–7322

  51. Nishimura SY, Vrljic M, Klein LO, McConnell HM, Moerner WE (2006) Cholesterol depletion induces solid-like regions in the plasma membrane. Biophys J 90:927–938

  52. Oesterhelt F, Oesterhelt D, Pfeiffer M, Engel A, Gaub HE, Muller DJ (2000) Unfolding pathways of individual bacteriorhodopsins. Science 288:143–146

  53. Ohsugi Y, Saito K, Tamura M, Kinjo M (2006) Lateral mobility of membrane-binding proteins in living cells measured by total internal reflection fluorescence correlation spectroscopy. Biophys J 91:3456–3464

  54. Orr G, Hu D, Ozcelik S, Opresko LK, Wiley HS, Colson SD (2005) Cholesterol dictates the freedom of EGF receptors and HER2 in the plane of the membrane. Biophys J 89:1362–1373

  55. Perez JB, Segura JM, Abankwa D, Piguet J, Martinez KL, Vogel H (2006) Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains. J Mol Biol 363:918–930

  56. Pfister G, Stroh CM, Perschinka H, Kind M, Knoflach M, Hinterdorfer P, Wick G (2005) Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy. J Cell Sci 118:1587–1594

  57. Puntheeranurak T, Wildling L, Gruber HJ, Kinne RK, Hinterdorfer P (2006) Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells. J Cell Sci 119:2960–2967

  58. Ries J, Schwille P (2006) Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy. Biophys J 91:1915–1924

  59. Ritchie K, Shan XY, Kondo J, Iwasawa K, Fujiwara T, Kusumi A (2005) Detection of non-Brownian diffusion in the cell membrane in single molecule tracking. Biophys J 88:2266–2277

  60. Robelek R, Lemker ES, Wiltschi B, Kirste V, Naumann R, Oesterhelt D, Sinner EK (2007) Incorporation of in vitro synthesized GPCR into a tethered artificial lipid membrane system. Angew Chem Int Ed Engl 46:605–608

  61. Sapra KT, Besir H, Oesterhelt D, Muller DJ (2006) Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy. J Mol Biol 355:640–650

  62. Schenk AD, Werten PJ, Scheuring S, de Groot BL, Muller SA, Stahlberg H, Philippsen A, Engel A (2005) The 4.5 A structure of human AQP2. J Mol Biol 350:278–289

  63. Scheuring S, Sturgis JN (2005) Chromatic adaptation of photosynthetic membranes. Science 309:484–487

  64. Scheuring S, Reiss-Husson F, Engel A, Rigaud JL, Ranck JL (2001) High-resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2. EMBO J 20:3029–3035

  65. Scheuring S, Seguin J, Marco S, Levy D, Breyton C, Robert B, Rigaud JL (2003a) AFM characterization of tilt and intrinsic flexibility of Rhodobacter sphaeroides light harvesting complex 2 (LH2). J Mol Biol 325:569–580

  66. Scheuring S, Seguin J, Marco S, Levy D, Robert B, Rigaud JL (2003b) Nanodissection and high-resolution imaging of the Rhodopseudomonas viridis photosynthetic core complex in native membranes by AFM. Atomic force microscopy. Proc Natl Acad Sci USA 100:1690–1693

  67. Scheuring S, Francia F, Busselez J, Melandri BA, Rigaud JL, Levy D (2004a) Structural role of PufX in the dimerization of the photosynthetic core complex of Rhodobacter sphaeroides. J Biol Chem 279:3620–3626

  68. Scheuring S, Rigaud JL, Sturgis JN (2004b) Variable LH2 stoichiometry and core clustering in native membranes of Rhodospirillum photometricum. EMBO J 23:4127–4133

  69. Scheuring S, Goncalves RP, Prima V, Sturgis JN (2006) The photosynthetic apparatus of Rhodopseudomonas palustris: structures and organization. J Mol Biol 358:83–96

  70. Schmidt T, Schutz GJ, Baumgartner W, Gruber HJ, Schindler H (1996) Imaging of single molecule diffusion. Proc Natl Acad Sci USA 93:2926–2929

  71. Schutz GJ, Schindler H, Schmidt T (1997) Single-molecule microscopy on model membranes reveals anomalous diffusion. Biophys J 73:1073–1080

  72. Schwille P (2001) Fluorescence correlation spectroscopy and its potential for intracellular applications. Cell Biochem Biophys 34:383–408

  73. Shibata SC, Hibino K, Mashimo T, Yanagida T, Sako Y (2006) Formation of signal transduction complexes during immobile phase of NGFR movements. Biochem Biophys Res Commun 342:316–322

  74. Suzuki K, Ritchie K, Kajikawa E, Fujiwara T, Kusumi A (2005) Rapid hop diffusion of a G-protein-coupled receptor in the plasma membrane as revealed by single-molecule techniques. Biophys J 88:3659–3680

  75. Tanuj SK, Park PS, Filipek S, Engel A, Muller DJ, Palczewski K (2006) Detecting molecular interactions that stabilize native bovine rhodopsin. J Mol Biol 358:255–269

  76. Teramura Y, Ichinose J, Takagi H, Nishida K, Yanagida T, Sako Y (2006) Single-molecule analysis of epidermal growth factor binding on the surface of living cells. EMBO J 25:4215–4222

  77. Uyemura T, Takagi H, Yanagida T, Sako Y (2005) Single-molecule analysis of epidermal growth factor signaling that leads to ultrasensitive calcium response. Biophys J 88:3720–3730

  78. van Meer G (2005) Cellular lipidomics. EMBO J 24:3159–3165

  79. Wawrezinieck L, Rigneault H, Marguet D, Lenne PF (2005) Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization. Biophys J 89:4029–4042

  80. Wenger J, Conchonaud F, Dintinger J, Wawrezinieck L, Ebbesen TW, Rigneault H, Marguet D, Lenne PF (2007) Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization. Biophys J 92:913–919

  81. Zimmerberg J, Gawrisch K (2006) The physical chemistry of biological membranes. Nat Chem Biol 2:564–567

  82. Zlatanova J, Lindsay SM, Leuba SH (2000) Single molecule force spectroscopy in biology using the atomic force microscope. Prog Biophys Mol Biol 74:37–61

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Aknowledgments

We thank J. Suckale, J. Ries, and S. Chiantia for careful reading. This work was supported by a Marie Curie Intra-European Fellowship (FP6) and German DGF (SCHW716/4-1).

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Correspondence to Petra Schwille.

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García-Sáez, A.J., Schwille, P. Single molecule techniques for the study of membrane proteins. Appl Microbiol Biotechnol 76, 257–266 (2007). https://doi.org/10.1007/s00253-007-1007-8

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Keywords

  • Membrane protein
  • Single molecule technique
  • Atomic force microscopy
  • Fluorescence correlation spectroscopy
  • Single particle tracking
  • Single molecule microscopy