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Biophysical Reviews

, Volume 10, Issue 2, pp 363–373 | Cite as

Hypothesis: structural heterogeneity of the unfolded proteins originating from the coupling of the local clusters and the long-range distance distribution

  • Satoshi Takahashi
  • Aya Yoshida
  • Hiroyuki Oikawa
Review

Abstract

We propose a hypothesis that explains two apparently contradicting observations for the heterogeneity of the unfolded proteins. First, the line confocal method of the single-molecule Förster resonance energy transfer (sm-FRET) spectroscopy revealed that the unfolded proteins possess broad peaks in the FRET efficiency plot, implying the significant heterogeneity that lasts longer than milliseconds. Second, the fluorescence correlation method demonstrated that the unfolded proteins fluctuate in the time scale shorter than 100 ns. To formulate the hypothesis, we first summarize the recent consensus for the structure and dynamics of the unfolded proteins. We next discuss the conventional method of the sm-FRET spectroscopy and its limitations for the analysis of the unfolded proteins, followed by the advantages of the line confocal method that revealed the heterogeneity. Finally, we propose that the structural heterogeneity formed by the local clustering of hydrophobic residues modulates the distribution of the long-range distance between the labeled chromophores, resulting in the broadening of the peak in the FRET efficiency plot. A clustering of hydrophobic residues around the chromophore might further contribute to the broadening. The proposed clusters are important for the understanding of protein folding mechanism.

Keywords

Sm-FRET spectroscopy Protein folding Dynamics and heterogeneity of the unfolded proteins 

Notes

Acknowledgements

We dedicate this article to Prof. Fumio Arisaka. We thank our collaborators and the former and current members of our laboratory.

Compliance with ethical standards

Funding

This work was supported by JSPS KAKENHI Grant Number JP25104007 (to S.T.) and JSPS KAKENHI Grant Number JP17K17608 (to H.O.).

Conflict of interest

Satoshi Takahashi declares that he has no conflict of interest. Aya Yoshida declares that she has no conflict of interest. Hiroyuki Oikawa declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Abaskharon RM, Gai F (2016) Meandering down the energy landscape of protein folding: are we there yet? Biophys J 110:1924–1932CrossRefPubMedPubMedCentralGoogle Scholar
  2. Aznauryan M, Delgado L, Soranno A, Nettels D, Huang J, Labhardt AM, Grzesiek S, Schuler B (2016) Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS. Proc Natl Acad Sci U S A 113:E5389–E5398CrossRefPubMedPubMedCentralGoogle Scholar
  3. Baxa MC, Haddadian EJ, Jumper JM, Freed KF, Sosnick TR (2014) Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations. Proc Natl Acad Sci U S A 111:15396–15401CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bernado P, Blanchard L, Timmins P, Marion D, Ruigrok RW, Blackledge M (2005) A structural model for unfolded proteins from residual dipolar couplings and small-angle x-ray scattering. Proc Natl Acad Sci U S A 102:17002–17007CrossRefPubMedPubMedCentralGoogle Scholar
  5. Borgia A, Wensley BG, Soranno A, Nettels D, Borgia MB, Hoffmann A, Pfeil SH, Lipman EA, Clarke J, Schuler B (2012) Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy. Nature Commun 3:1195 (9 pages)CrossRefGoogle Scholar
  6. Borgia A, Zheng W, Buholzer K, Borgia MB, Schüler A, Hofmann H, Soranno A, Nettels D, Gast K, Grishaev A, Best RB, Schuler B (2016) Consistent view of polypeptide chain expansion in chemical denaturants from multiple experimental methods. J Am Chem Soc 138:11714–11726CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bowler BE (2012) Residual structure in unfolded proteins. Curr Opin Struct Biol 22:4–13CrossRefPubMedGoogle Scholar
  8. Buckler DR, Haas E, Scheraga HA (1995) Analysis of the structure of ribonuclease A in native and partially denatured states by time-resolved noradiative dynamic excitation energy transfer between site-specific extrinsic probes. Biochemistry 34:15965–15978CrossRefPubMedGoogle Scholar
  9. Buscaglia M, Lapidus LJ, Eaton WA, Hofrichter J (2006) Effects of denaturants on the dynamics of loop formation in polypeptides. Biophys J 91:276–288CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chen H, Ahsan SS, Santiago-Berrios MB, Abruña HD, Webb WW (2010) Mechanisms of quenching of Alexa fluorophores by natural amino acids. J Am Chem Soc 132:7244–7245CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cho J-H, Meng W, Sato S, Kim EY, Schindelin H, Raleigh DP (2014) Energetically significant networks of coupled interactions within an unfolded protein. Proc Natl Acad Sci U S A 111:12079–12084CrossRefPubMedPubMedCentralGoogle Scholar
  12. Dar TA, Schaeffer RD, Daggett V, Bowler BE (2011) Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c’. Biochemistry 50:1029–1041CrossRefPubMedPubMedCentralGoogle Scholar
  13. Deniz AA, Dahan M, Grunwell JR, Ha T, Faulhaber AE, Chemla DS, Weiss S, Schultz PG (1999) Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations. Proc Natl Acad Sci U S A 96:3670–3675CrossRefPubMedPubMedCentralGoogle Scholar
  14. Deniz AA, Laurence TA, Beligere GS, Dahan M, Martin AB, Chemla DS, Dawson PE, Schultz PG, Weiss S (2000) Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2. Proc Natl Acad Sci U S A 97:5179–5184CrossRefPubMedPubMedCentralGoogle Scholar
  15. Dill KA, MacCallum JL (2012) The protein-folding problem, 50 years on. Science 338:1042–1046CrossRefPubMedGoogle Scholar
  16. Felitsky D, Lietzow MA, Dyson HJ, Wright PE (2008) Modeling transient collapsed states of an unfolded protein to provide insights into early folding events. Proc Natl Acad Sci U S A 105:6278–6283CrossRefPubMedPubMedCentralGoogle Scholar
  17. Fitzkee NC, Rose GD (2004) Reassessing random-coil statistics in unfolded proteins. Proc Natl Acad Sci U S A 101:12497–12502CrossRefPubMedPubMedCentralGoogle Scholar
  18. Fuertes G, Banterle N, Ruff KM, Chowdhury A, Mercadante D, Koehler C, Kachala M, Estrada Girona G, Milles S, Mishra A, Onck PR, Gräter F, Esteban-Martín S, Pappu RV, Svergun DI, Lemke EA (2017) Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs FRET measurements. Proc Natl Acad Sci USA 114:E6342–E6351CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hagen SJ, Hofrichter J, Szabo A, Eaton WA (1996) Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding. Proc Natl Acad Sci U S A 93:11615–11617CrossRefPubMedPubMedCentralGoogle Scholar
  20. Haran G (2012) How, when and why proteins collapse: the relation to folding. Curr Opin Struct Biol 22:14–20CrossRefPubMedGoogle Scholar
  21. Hoefling M, Lima N, Haenni D, Seidel CAM, Schuler B, Grubmüller H (2011) Structural heterogeneity and quantitative FRET efficiency distributions of polyprolines through a hybrid atomistic simulation and Monte Carlo approach. PLoS One 6:e19791 (19 pages)CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hoffmann H (2016) Understanding disordered and unfolded proteins using single-molecule FRET and polymer theory. Methods Appl Fluoresc 4:042003 (8 pages)CrossRefGoogle Scholar
  23. Hofmann H, Soranno A, Borgia A, Gast K, Nettels D, Schuler B (2012) Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy. Proc Natl Acad Sci U S A 109:16155–16160CrossRefPubMedPubMedCentralGoogle Scholar
  24. Holden SJ, Uphoff S, Hohlbein J, Yadin D, Le Reste L, Britton OJ, Kapanidis AN (2010) Defining the limits of single-molecule FRET resolution in TIRF microscopy. Biophys J 99:3102–3111CrossRefPubMedPubMedCentralGoogle Scholar
  25. Huang J-r, Grzesiek S (2010) Ensemble calculations of unstructured proteins constrained by RDC and PRE data: a case study of urea-denatured ubiquitin. J Am Chem Soc 132:694–705CrossRefPubMedGoogle Scholar
  26. Jensen MR, Zweckstetter M, Huang J-r, Blackledge M (2014) Exploring free-energy landscapes of intrinsically disordered proteins at atomic resolution using NMR spectroscopy. Chem Rev 114:6632–6660CrossRefPubMedGoogle Scholar
  27. Jha AK, Colubri A, Freed KF, Sosnick TR (2005) Statistical coil model of the unfolded state: resolving the reconciliation problem. Proc Natl Acad Sci U S A 102:13099–13104CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kalinin S, Sisamakis E, Magennis SW, Felekyan S, Seidel CAM (2010) On the origin of broadening of single-molecule FRET efficiency distributions beyond shot noise limits. J Phys Chem B 114:6197–6206CrossRefPubMedGoogle Scholar
  29. Klein-Seetharaman J, Oikawa M, Grimshaw SB, Wirmer J, Duchardt E, Ueda T, Imoto T, Smith LJ, Dobson CM, Schwalbe H (2002) Long-range interactions within a nonnative protein. Science 295:1719–1722CrossRefPubMedGoogle Scholar
  30. Kohn JE, Millett IS, Jacob J, Zagrovic B, Dillon TM, Cingel N, Dothager RS, Seifert S, Thiyagarajan P, Sosnick TR, Hasan MZ, Pande VS, Ruczinski I, Doniach S, Plaxco KW. (2004) Random-coil behavior and the dimensions of chemically unfolded proteins. Proc. Natl. Acad. Sci. USA. 101, 12491–12496. Erratum in: (2005) Proc. Natl. Acad. Sci. USA. 102, 14475Google Scholar
  31. Konuma T, Kimura T, Matsumoto S, Goto Y, Fujisawa T, Fersht AR, Takahashi S (2011) Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase. J Mol Biol 405:1284–1294CrossRefPubMedGoogle Scholar
  32. Kuzmenkina EV, Heyes CD, Nienhaus GU (2005) Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions. Proc Natl Acad Sci U S A 102:15471–15476CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lapidus LJ, Eaton WA, Hofrichter J (2000) Measuring the rate of intramolecular contact formation in polypeptides. Proc Natl Acad Sci U S A 97:7220–7225CrossRefPubMedPubMedCentralGoogle Scholar
  34. Maity H, Reddy G (2016) Folding of protein L with implications for collapse in the denatured state ensemble. J Am Chem Soc 138:2609–2616CrossRefPubMedGoogle Scholar
  35. Meier S, Grzesiek S, Blackledge M (2007) Direct observation of dipolar couplings and hydrogen bonds across a β-hairpin in 8 M urea. J Am Chem Soc 129:9799–9807CrossRefPubMedGoogle Scholar
  36. Meng W, Luan B, Lyle N, Pappu RV, Raleigh DP (2013) The denatured state ensemble contains significant local and long-range structure under native conditions: analysis of the N-terminal domain of ribosomal protein L9. Biochemistry 52:2662–2671CrossRefPubMedGoogle Scholar
  37. 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 U S A 104:1528–1533CrossRefPubMedPubMedCentralGoogle Scholar
  38. Moglich A, Joder K, Kiefhaber T (2006) End-to-end distance distributions and intrachain diffusion constants in unfolded polypeptide chains indicate intramolecular hydrogen bond formation. Proc. Natl. Acad. Sci. USA. 103, 12394–12399. Erratum in: (2008) Proc. Natl. Acad. Sci. USA. 105, 6787Google Scholar
  39. Nettels D, Gopich IV, Hoffmann A, Schuler B (2007) Ultrafast dynamics of protein collapse from single-molecule photon statistics. Proc Natl Acad Sci U S A 104:2655–2660CrossRefPubMedPubMedCentralGoogle Scholar
  40. Ohnishi S, Lee AL, Edgell MH, Shortle D (2004) Direct demonstration of structural similarity between native and denatured eglin C. Biochemistry 43:4064–4070CrossRefPubMedGoogle Scholar
  41. Oikawa H, Suzuki Y, Saito M, Kamagata K, Arai M, Takahashi S (2013) Microsecond dynamics of an unfolded protein by a line confocal tracking of single molecule fluorescence. Sci Rep 3:2151CrossRefPubMedPubMedCentralGoogle Scholar
  42. Oikawa H, Kamagata K, Arai M, Takahashi S (2015) Complexity of the folding transition of the B domain of protein A revealed by the high-speed tracking of single-molecule fluorescence time series. J Phys Chem B 119:6081–6091CrossRefPubMedGoogle Scholar
  43. Oikawa H, Takahashi T, Kamonprasertsuk S, Takahashi S (2018) Microsecond resolved single-molecule FRET time series measurements based on line confocal optical system combined with hybrid photodetectors. Phys Chem Chem Phys 20:3277–3285CrossRefPubMedGoogle Scholar
  44. Plaxco KW, Millett IS, Segel DJ, Doniach S, Baker D (1999) Chain collapse can occur concomitantly with the rate-limiting step in protein folding. Nat Struct Biol 6:554–556CrossRefPubMedGoogle Scholar
  45. Riback JA, Bowman MA, Zmyslowski AM, Knoverek CR, Jumper JM, Hinshaw JR, Kaye EB, Freed KF, Clark PL, Sosnick TR (2017) Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water. Science 358:238–241CrossRefPubMedGoogle Scholar
  46. Saito M, Kamonprasertsuk S, Suzuki S, Nanatani K, Oikawa H, Kushiro K, Takai M, Chen PT, Chen EH, Chen RP, Takahashi S (2016) Significant heterogeneity and slow dynamics of the unfolded ubiquitin detected by the line confocal method of single-molecule fluorescence spectroscopy. J Phys Chem B 120:8818–8829CrossRefPubMedGoogle Scholar
  47. Sakurai K, Yagi M, Konuma T, Takahashi S, Nishimura C, Goto Y (2017) Non-native α-helices in the initial folding intermediate facilitate the ordered assembly of the β-barrel in β-lactoglobulin. Biochemistry 56:4799–4807CrossRefPubMedGoogle Scholar
  48. Schuler B, Lipman EA, Eaton WA (2002) Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy. Nature 419:743–747CrossRefPubMedGoogle Scholar
  49. Schuler B, Soranno A, Hofmann H, Nettels D (2016) Single-molecule FRET spectroscopy and the polymer physics of unfolded and intrinsically disordered proteins. Annu Rev Biophys 45:207–231CrossRefPubMedGoogle Scholar
  50. Sherman E, Haran G (2006) Coil-globule transition in the denatured state of a small protein. Proc Natl Acad Sci U S A 103:11539–11543CrossRefPubMedPubMedCentralGoogle Scholar
  51. Shortle D (2002) The expanded denatured state: an ensemble of conformations trapped in a locally encoded topological space. Adv Protein Chem 62:1–23CrossRefPubMedGoogle Scholar
  52. Shortle D, Ackerman MS (2001) Persistence of native-like topology in a denatured protein in 8 M urea. Science 293:487–489CrossRefPubMedGoogle Scholar
  53. Song J, Gomes G-N, Shi T, Gradinaru CC, Chan HS (2017) Conformational heterogeneity and FRET data interpretation for dimensions of unfolded proteins. Biophys J 113:1012–1024CrossRefPubMedGoogle Scholar
  54. Soranno A, Longhi R, Bellini T, Buscaglia M (2009) Kinetics of contact formation and end-to-end distance distributions of swollen disordered peptides. Biophys J 96:1515–1528CrossRefPubMedPubMedCentralGoogle Scholar
  55. Soranno A, Buchli B, Nettels D, Cheng RR, Müller-Späth S, Pfeil SH, Hoffmann A, Lipman EA, Makarov DE, Schuler B (2012) Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy. Proc Natl Acad Sci U S A 109:17800–17806CrossRefPubMedPubMedCentralGoogle Scholar
  56. Soranno A, Holla A, Dingfelder F, Nettels D, Makarov DE, Schuler B (2017) Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations. Proc Natl Acad Sci U S A 114:E1833–E1839CrossRefPubMedPubMedCentralGoogle Scholar
  57. Sosnick TR, Barrick D (2011) The folding of single domain proteins: have we reached a consensus? Curr Opin Struct Biol 21:12–24CrossRefPubMedGoogle Scholar
  58. Sziegat F, Silvers R, Hähnke M, Jensen MR, Blackledge M, Wirmer-Bartoschek J, Schwalbe H (2012) Disentangling the coil: modulation of conformational and dynamic properties by site-directed mutation in the non-native state of hen egg white lysozyme. Biochemistry 51:3361–3372CrossRefPubMedGoogle Scholar
  59. Takahashi S, Kamagata K, Oikawa H (2016) Where the complex things are: single molecule and ensemble spectroscopic investigations of protein folding dynamics. Curr Opin Struct Biol 36:1–9CrossRefPubMedGoogle Scholar
  60. Tezuka-Kawakami T, Gell C, Brockwell DJ, Radford SE, Smith DA (2006) Urea-induced unfolding of the immunity protein Im9 monitored by spFRET. Biophys J 91:L42–L44CrossRefPubMedPubMedCentralGoogle Scholar
  61. Udgaonkar JB (2013) Polypeptide chain collapse and protein folding. Arch Biochem Biophys 531:24–33CrossRefPubMedGoogle Scholar
  62. Waldauer SA, Bakajin O, Lapidus LJ (2010) Extremely slow intramolecular diffusion in unfolded protein L. Proc Natl Acad Sci U S A 107:13713–13717CrossRefPubMedPubMedCentralGoogle Scholar
  63. Watkins HM, Simon AJ, Sosnick TR, Lipman EA, Hjelm RP, Plaxco KW (2015) Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions. Proc Natl Acad Sci U S A 112:6631–6636CrossRefPubMedPubMedCentralGoogle Scholar
  64. Xue Y, Skrynnikov NR (2011) Motion of a disordered polypeptide chain as studied by paramagnetic relaxation enhancements, 15N relaxation, and molecular dynamics simulations: how fast is segmental diffusion in denatured ubiquitin? J Am Chem Soc 133:14614–14628CrossRefPubMedGoogle Scholar
  65. Yoo TY, Meisburger SP, Hinshaw J, Pollack L, Haran G, Sosnick TR, Plaxco K (2012) Small-angle X-ray scattering and single-molecule FRET spectroscopy produce highly divergent views of the low-denaturant unfolded state. J Mol Biol 418:226–236CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Multidisciplinary Research for Advanced MaterialsTohoku UniversitySendaiJapan
  2. 2.Department of Chemistry, Graduate school of ScienceTohoku UniversitySendaiJapan

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