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Macromolecular Models by Single Molecule FRET

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Biophysics and Structure to Counter Threats and Challenges

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Abstract

Single molecule fluorescence energy transfer (FRET) experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined by docking calculations. Here we discuss recent applications of single molecule FRET to obtain a model of the synaptotagmin-1:SNARE complex and to study the conformation of PSD-95.

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References

  1. Amir D, Haas E (1987) Estimation of intramolecular distance distributions in bovine pancreatic trypsin inhibitor by site-specific labeling and nonradiative excitation energy-transfer measurements. Biochemistry 26:2162–2175

    Article  Google Scholar 

  2. Amir D, Haas E (1988) Reduced bovine pancreatic trypsin inhibitor has a compact structure. Biochemistry 27:8889–8893

    Article  Google Scholar 

  3. Bhattacharyya RP, Remenyi A, Yeh BJ, Lim WA (2006) Domains, motifs, and scaffolds: the role of modular interactions in the evolution and wiring of cell signaling circuits. Annu Rev Biochem 75:655–680

    Article  Google Scholar 

  4. Brunger AT (1992) X-PLOR, version 3.1. A system X-ray crystallograpjy and NMR. Yale University Press, New Haven

    Google Scholar 

  5. Brunger AT (2005) Structure and function of SNARE and SNARE-interacting proteins. Q Rev Biophys 38:1–47

    Article  Google Scholar 

  6. Brunger AT, Nilges M (1993) Computational challenges for macromolecular structure determination by X-ray crystallography and solution NMR-spectroscopy. Q Rev Biophys 26:49–125

    Article  Google Scholar 

  7. Brunger AT, Strop P, Vrljic M, Chu S, Weninger KR (2011) Three-dimensional molecular modeling with single molecule FRET. J Struct Biol 173:497–505

    Article  Google Scholar 

  8. Cherny DI, Eperon IC, Bagshaw CR (2009) Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes. Eur Biophys J 38:395–405

    Article  Google Scholar 

  9. Cho W, Stahelin RV (2006) Membrane binding and subcellular targeting of C2 domains. Biochim Biophys Acta 1761:838–849

    Article  Google Scholar 

  10. Cho KO, Hunt CA, Kennedy MB (1992) The rat brain postsynaptic density fraction contains a homolog of the drosophila discs-large tumor suppressor protein. Neuron 9:929–942

    Article  Google Scholar 

  11. Choi UB, Strop P, Vrljic M, Chu S, Brunger AT, Weninger KR (2010) Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex. Nat Struct Mol Biol 17:318–324

    Article  Google Scholar 

  12. Chung HS, Louis JM, Eaton WA (2009) Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories. Proc Natl Acad Sci U S A 106:11837–11844

    Article  ADS  Google Scholar 

  13. Dahan M, Deniz AA, Ha T, Chemla DS, Schultz PG, Weiss S (1999) Ratiometric measurement and identification of single diffusing molecules. Chem Phys 247:85–106

    Article  ADS  Google Scholar 

  14. Dave R, Terry DS, Munro JB, Blanchard SC (2009) Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging. Biophys J 96:2371–2381

    Article  Google Scholar 

  15. de Vries SJ, van Dijk AD, Krzeminski M, van Dijk M, Thureau A, Hsu V, Wassenaar T, Bonvin AM (2007) HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets. Proteins 69:726–733

    Article  Google Scholar 

  16. de Wit H, Walter AM, Milosevic I, Gulyas-Kovacs A, Riedel D, Sorensen JB, Verhage M (2009) Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes. Cell 138:935–946

    Article  Google Scholar 

  17. Deniz AA, Mukhopadhyay S, Lemke EA (2008) Single-molecule biophysics: at the interface of biology, physics and chemistry. J R Soc Interface 5:15–45

    Article  Google Scholar 

  18. DeRocco V, Anderson T, Piehler J, Erie DA, Weninger K (2010) Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes. Biotechniques 49:807–816

    Article  Google Scholar 

  19. Dominguez C, Boelens R, Bonvin AM (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731–1737

    Article  Google Scholar 

  20. Engh RA, Huber R (1991) Accurate bond and angle parameters for X-Ray protein- structure refinement. Acta Crystallogr A 47:392–400

    Article  Google Scholar 

  21. Feng W, Shi Y, Li M, Zhang M (2003) Tandem PDZ repeats in glutamate receptor-interacting proteins have a novel mode of PDZ domain-mediated target binding. Nat Struct Biol 10:972–978

    Article  Google Scholar 

  22. Fernandez-Chacon R, Konigstorfer A, Gerber SH, Garcia J, Matos MF, Stevens CF, Brose N, Rizo J, Rosenmund C, Sãdhof TC (2001) Synaptotagmin I functions as a calcium regulator of release probability. Nature 410:41–49

    Article  ADS  Google Scholar 

  23. Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys 2:55–57

    Article  MATH  Google Scholar 

  24. Fuson KL, Montes M, Robert JJ, Sutton RB (2007) Structure of human synaptotagmin 1 C2AB in the absence of Ca2+ reveals a novel domain association. Biochemistry 46:13041–13048

    Article  Google Scholar 

  25. Giraudo CG, Garcia-Diaz A, Eng WS, Chen Y, Hendrickson WA, Melia TJ, Rothman JE (2009) Alternative zippering as an on-off switch for SNARE-mediated fusion. Science 323:512–516

    Article  ADS  Google Scholar 

  26. Goult BT, Rapley JD, Dart C, Kitmitto A, Grossmann JG, Leyland ML, Lian LY (2007) Small-angle X-ray scattering and NMR studies of the conformation of the PDZ region of SAP97 and its interactions with Kir2.1. Biochemistry 46:14117–14128

    Article  Google Scholar 

  27. Ha T, Ting AY, Liang J, Caldwell WB, Deniz AA, Chemla DS, Schultz PG, Weiss S (1999) Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc Natl Acad Sci USA 96:893–898

    Article  ADS  Google Scholar 

  28. Ha T, Ting AY, Liang J, Deniz AA, Chemla DS, Schultz PG, Weiss S (1999) Temporal fluctuations of fluorescence resonance energy transfer between two dyes conjugated to a single protein. Chem Phys 247:107–118

    Article  ADS  Google Scholar 

  29. Harris BZ, Lim WA (2001) Mechanism and role of PDZ domains in signaling complex assembly. J Cell Sci 114:3219–3231

    Google Scholar 

  30. Haugland RP (2005) The handbook: A guide to fluorescent probes and labeling technologies. Molecular Probes, Carlsbad

    Google Scholar 

  31. Hung AY, Sheng M (2002) PDZ domains: structural modules for protein complex assembly. J Biol Chem 277:5699–5702

    Article  Google Scholar 

  32. Iqbal A, Arslan S, Okumus B, Wilson TJ, Giraud G, Norman DG, Ha T, Lilley DM (2008) Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids. Proc Natl Acad Sci U S A 105:11176–11181

    Article  ADS  Google Scholar 

  33. Irie M, Hata Y, Takeuchi M, Ichtchenko K, Toyoda A, Hirao K, Takai Y, Rosahl TW, Sudhof TC (1997) Binding of neuroligins to PSD-95. Science 277:1511–1515

    Article  Google Scholar 

  34. Kang BS, Cooper DR, Jelen F, Devedjiev Y, Derewenda U, Dauter Z, Otlewski J, Derewenda ZS (2003) PDZ tandem of human syntenin: crystal structure and functional properties. Structure 11:459–468

    Article  Google Scholar 

  35. Lee NK, Kapanidis AN, Wang Y, Michalet X, Mukhopadhyay J, Ebright RH, Weiss S (2005) Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. Biophys J 88:2939–2953

    Article  Google Scholar 

  36. Lee J, Lee S, Ragunathan K, Joo C, Ha T, Hohng S (2010) Single-molecule four-color FRET. Angew Chem Int Ed Engl 49:9922–9925

    Article  Google Scholar 

  37. Long JF, Tochio H, Wang P, Fan JS, Sala C, Niethammer M, Sheng M, Zhang M (2003) Supramodular structure and synergistic target binding of the N-terminal tandem PDZ domains of PSD-95. J Mol Biol 327:203–214

    Article  Google Scholar 

  38. Long JF, Feng W, Wang R, Chan LN, Ip FC, Xia J, Ip NY, Zhang M (2005) Autoinhibition of X11/Mint scaffold proteins revealed by the closed conformation of the PDZ tandem. Nat Struct Mol Biol 12:722–728

    Article  Google Scholar 

  39. Long J, Wei Z, Feng W, Yu C, Zhao YX, Zhang M (2008) Supramodular nature of GRIP1 revealed by the structure of its PDZ12 tandem in complex with the carboxyl tail of Fras1. J Mol Biol 375:1457–1468

    Article  Google Scholar 

  40. Maximov A, Tang J, Yang X, Pang ZP, Sudhof TC (2009) Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 323:516–521

    Article  ADS  Google Scholar 

  41. McCann J, Choi UB, Zheng L, Weninger K, Bowen ME (2010) Optimizing methods to recover absolute FRET efficiency from immobilized single molecules. Biophys J 99:961–970

    Article  Google Scholar 

  42. McCann J, Zheng L, Chiantia S, Bowen ME (2011) Domain orientation in the tandem PDZ supramodule from PSD-95 is maintained in the full-length protein. Structure 19:810–820

    Article  Google Scholar 

  43. Merchant KA, Best RB, Louis JM, Gopich IV, Eaton WA (2007) Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations. Proc Natl Acad Sci 104:1528–1533

    Article  ADS  Google Scholar 

  44. Nir E, Michalet X, Hamadani KM, Laurence TA, Neuhauser D, Kovchegov Y, Weiss S (2006) Shot-noise limited single-molecule FRET histograms: comparison between theory and experiments. J Phys Chem B 110:22103–22124

    Article  Google Scholar 

  45. Pang ZP, Shin OH, Meyer AC, Rosenmund C, Sudhof TC (2006) A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2 + −dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis. J Neurosci 26:12556–12565

    Article  Google Scholar 

  46. Pawson T, Scott JD (1997) Signaling through scaffold, anchoring, and adaptor proteins. Science 278:2075–2080

    Article  ADS  Google Scholar 

  47. Peterson FC, Penkert RR, Volkman BF, Prehoda KE (2004) Cdc42 regulates the Par-6 PDZ domain through an allosteric CRIB-PDZ transition. Mol Cell 13:665–676

    Article  Google Scholar 

  48. Piserchio A, Pellegrini M, Mehta S, Blackman SM, Garcia EP, Marshall J, Mierke DF (2002) The PDZ1 domain of SAP90. Characterization of structure and binding. J Biol Chem 277:6967–6973

    Article  Google Scholar 

  49. Rasnik I, Myong S, Cheng W, Lohman TM, Ha T (2004) DNA-binding orientation and domain conformation of the E. coli rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes. J Mol Biol 336:395–408

    Article  Google Scholar 

  50. Rasnik I, McKinney SA, Ha T (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3:891–893

    Article  Google Scholar 

  51. Rhee JS, Li LY, Shin OH, Rah JC, Rizo J, Sudhof TC, Rosenmund C (2005) Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1. Proc Natl Acad Sci U S A 102:18664–18669

    Article  ADS  Google Scholar 

  52. Rice LM, Brunger AT (1994) Torsion angle dynamics: reduced variable conformational sampling enhances crystallographic structure refinement. Proteins 19:277–290

    Article  Google Scholar 

  53. Rickman C, Jimenez JL, Graham ME, Archer DA, Soloviev M, Burgoyne RD, Davletov B (2006) Conserved prefusion protein assembly in regulated exocytosis. Mol Biol Cell 17:283–294

    Article  Google Scholar 

  54. Rizo J, Rosenmund C (2008) Synaptic vesicle fusion. Nat Struct Mol Biol 15:665–674

    Article  Google Scholar 

  55. Rothwell PJ, Berger S, Kensch O, Felekyan S, Antonik M, Wöhrl BM, Restle T, Goody RS, Seidel CAM (2003) Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes. Proc Natl Acad Sci U S A 100:1655–1660

    Article  ADS  Google Scholar 

  56. Roy R, Kozlov AG, Lohman TM, Ha T (2007) Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein. J Mol Biol 369:1244–1257

    Article  Google Scholar 

  57. Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5:507–516

    Article  Google Scholar 

  58. Sainlos M, Tigaret C, Poujol C, Olivier NB, Bard L, Breillat C, Thiolon K, Choquet D, Imperiali B (2010) Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization. Nat Chem Biol 7:81–91

    Article  Google Scholar 

  59. Sakon JJ, Weninger KR (2010) Detecting the conformation of individual proteins in live cells. Nat Methods 7:203–205

    Article  Google Scholar 

  60. Schroder GF, Levitt M, Brunger AT (2010) Super-resolution biomolecular crystallography with low-resolution data. Nature 464:1218–1222

    Article  ADS  Google Scholar 

  61. Schwieters CD, Clore GM (2001) Internal coordinates for molecular dynamics and minimization in structure determination and refinement. J Magn Reson 152:288–302

    Article  ADS  Google Scholar 

  62. Stryer L, Haugland RP (1967) Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci USA 58:719–726

    Article  ADS  Google Scholar 

  63. Sutton RB, Fasshauer D, Jahn R, Brunger AT (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395:347–353

    Article  ADS  Google Scholar 

  64. Tochio H, Hung F, Li M, Bredt DS, Zhang M (2000) Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density-95. J Mol Biol 295:225–237

    Article  Google Scholar 

  65. van den Berk LC, Landi E, Walma T, Vuister GW, Dente L, Hendriks WJ (2007) An allosteric intramolecular PDZ-PDZ interaction modulates PTP-BL PDZ2 binding specificity. Biochemistry 46:13629–13637

    Article  Google Scholar 

  66. Vrljic M, Strop P, Ernst JA, Sutton RB, Chu S, Brunger AT (2010) Molecular mechanism of the synaptotagmin-SNARE interaction in Ca2+-triggered vesicle fusion. Nat Struct Mol Biol 17:325–331

    Article  Google Scholar 

  67. Watkins LP, Chang H, Yang H (2006) Quantitative single-molecule conformational distributions: a case study with poly-(L-proline). J Phys Chem A 110:5191–5203

    Article  Google Scholar 

  68. Weninger K, Bowen ME, Chu S, Brunger AT (2003) Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations. Proc Natl Acad Sci USA 100:14800–14805

    Article  ADS  Google Scholar 

  69. Xue M, Ma C, Craig TK, Rosenmund C, Rizo J (2008) The Janus-faced nature of the C(2)B domain is fundamental for synaptotagmin-1 function. Nat Struct Mol Biol 15:1160–1168

    Article  Google Scholar 

  70. Zhang Q, Fan JS, Zhang M (2001) Interdomain chaperoning between PSD-95, Dlg, and Zo-1 (PDZ) domains of glutamate receptor-interacting proteins. J Biol Chem 276:43216–43220

    Article  Google Scholar 

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Acknowledgments

We thank the National Institutes of Health for support (to A.T.B., RO1-MH63105), and a Career Award at the Scientific Interface from the Burroughs Wellcome Fund (to K.W.). Part of this material has been published in modified form in the Journal of Structural Biology [7].

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Authors and Affiliations

  1. The Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science, Stanford University, James H. Clark Center, Room E300-C, Stanford, CA, 94305, USA

    Axel T. Brunger, Pavel Strop & Marija Vrljic

  2. Department of Physiology and Biophysics, Stony Brook University Medical Center, Stony Brook, NY, 11794-8661, USA

    Mark Bowen

  3. Formerly Lawrence Berkeley National Laboratory and Physics and Cell Biology Departments, University of California at Berkeley, Berkeley, CA, 94720, USA

    Steven Chu

  4. Department of Physics, North Carolina State University, Raleigh, NC, 27695, USA

    Keith R. Weninger

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  1. Axel T. Brunger
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  2. Pavel Strop
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  3. Marija Vrljic
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  4. Mark Bowen
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  5. Steven Chu
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  6. Keith R. Weninger
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Correspondence to Axel T. Brunger or Keith R. Weninger .

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Editors and Affiliations

  1. , Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, D105a Fairchild Sciences Building, Stanford, 94305-5126, USA

    Joseph D. Puglisi

  2. , Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Fairchild Science Building D100E, Stanford, 94305-5126, USA

    Manolia V. Margaris

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Brunger, A.T., Strop, P., Vrljic, M., Bowen, M., Chu, S., Weninger, K.R. (2012). Macromolecular Models by Single Molecule FRET. In: Puglisi, J., Margaris, M. (eds) Biophysics and Structure to Counter Threats and Challenges. NATO Science for Peace and Security Series B: Physics and Biophysics. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-4923-8_1

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