Biophysical Reviews

, Volume 5, Issue 2, pp 85–98 | Cite as

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

  • Tomer Orevi
  • Gil Rahamim
  • Gershon Hazan
  • Dan Amir
  • Elisha Haas


The extremely fast and efficient folding transition (in seconds) of globular proteins led to the search for some unifying principles embedded in the physics of the folding polypeptides. Most of the proposed mechanisms highlight the role of local interactions that stabilize secondary structure elements or a folding nucleus as the starting point of the folding pathways, i.e., a “bottom–up” mechanism. Non-local interactions were assumed either to stabilize the nucleus or lead to the later steps of coalescence of the secondary structure elements. An alternative mechanism was proposed, an “up–down” mechanism in which it was assumed that folding starts with the formation of very few non-local interactions which form closed long loops at the initiation of folding. The possible biological advantage of this mechanism, the “loop hypothesis”, is that the hydrophobic collapse is associated with ordered compactization which reduces the chance for degradation and misfolding. In the present review the experiments, simulations and theoretical consideration that either directly or indirectly support this mechanism are summarized. It is argued that experiments monitoring the time-dependent development of the formation of specifically targeted early-formed sub-domain structural elements, either long loops or secondary structure elements, are necessary. This can be achieved by the time-resolved FRET-based “double kinetics” method in combination with mutational studies. Yet, attempts to improve the time resolution of the folding initiation should be extended down to the sub-microsecond time regime in order to design experiments that would resolve the classes of proteins which first fold by local or non-local interactions.


Protein folding Loop hypothesis Hydrophobic collapse Ordered compatization 



We are grateful to Mr. E. Zimerman and D. Freedman for excellent technical assistance and to Eldad Ben Ishay, Eitan Lerner, and Asaf Grupi for their contributions and discussions. This study was supported by grants from the Israel Science Foundation (ISF1464/10); the EU Marie Curie TOK grant (29936), the US-Israel Binational science foundation (BSF 2005270); the Damadian Center for Magnetic Resonance Research, Bar-Ilan University.

Conflict of interest


Supplementary material

12551_2013_113_MOESM1_ESM.docx (40 kb)
ESM 1 (DOCX 39 kb)


  1. Abkevich VI, Gutin AM et al (1994) Specific nucleus as the transition state for protein folding: evidence from the lattice model. Biochemistry 33(33):10026–10036PubMedGoogle Scholar
  2. Abkevich VI, Gutin AM et al (1995) Impact of local and non-local interactions on thermodynamics and kinetics of protein folding. J Mol Biol 252(4):460–471PubMedGoogle Scholar
  3. Anfinsen CB, Scheraga HA (1975) Experimental and theoretical aspects of protein folding. Adv Protein Chem 29:205–300PubMedGoogle Scholar
  4. Anil B, Sato S et al (2005) Fine structure analysis of a protein folding transition state; distinguishing between hydrophobic stabilization and specific packing. J Mol Biol 354(3):693–705PubMedGoogle Scholar
  5. Aurora R, Creamer TP et al (1997) Local interactions in protein folding: lessons from the alpha-helix. J Biol Chem 272(3):1413–1416PubMedGoogle Scholar
  6. Bai Y, Sosnick TR et al (1995) Protein folding intermediates: native-state hydrogen exchange. Science 269(5221):192–197PubMedGoogle Scholar
  7. Baldwin RL, Rose GD (1999a) Is protein folding hierarchic? I. Local structure and peptide folding. Trends Biochem Sci 24(1):26–33PubMedGoogle Scholar
  8. Baldwin RL, Rose GD (1999b) Is protein folding hierarchic? II. Folding intermediates and transition states. Trends Biochem Sci 24(2):77–83PubMedGoogle Scholar
  9. Beechem JM, Haas E (1989) Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements. Biophys J 55(6):1225–1236PubMedGoogle Scholar
  10. Ben Ishay E, Hazan G, et al (2012a) An instrument for fast acquisition of fluorescence decay curves at picosecond resolution designed for “double kinetics” experiments: Application to FRET study of protein folding. Rev Sci Instruments 83(8):084301Google Scholar
  11. Ben Ishay E, Rahamim G et al (2012b) Fast subdomain folding prior to the global refolding transition of E. coli adenylate kinase: a double kinetics study. J Mol Biol 423(4):613–623PubMedGoogle Scholar
  12. Berezovsky IN, Kirzhner VM et al (2002) Closed loops: persistence of the protein chain returns. Protein Eng 15(12):955–957PubMedGoogle Scholar
  13. Berezovsky IN, Trifonov EN (2001) Loop fold nature of globular proteins. Protein Eng 14(6):403–407PubMedGoogle Scholar
  14. Berezovsky IN, Trifonov EN (2002) Flowering buds of globular proteins: transpiring simplicity of protein organization. Comp Funct Genomics 3(6):525–534PubMedGoogle Scholar
  15. Bilsel O, Matthews CR (2006) Molecular dimensions and their distributions in early folding intermediates. Curr Opin Struct Biol 16(1):86–93PubMedGoogle Scholar
  16. Bolen DW, Rose GD (2008) Structure and energetics of the hydrogen-bonded backbone in protein folding. Annu Rev Biochem 77:339–362PubMedGoogle Scholar
  17. Bowie JU, Eisenberg D (1994) An evolutionary approach to folding small alpha-helical proteins that uses sequence information and an empirical guiding fitness function. Proc Natl Acad Sci USA 91(10):4436–4440PubMedGoogle Scholar
  18. Buscaglia M, Schuler B et al (2003) Kinetics of intramolecular contact formation in a denatured protein. J Mol Biol 332(1):9–12PubMedGoogle Scholar
  19. Camacho CJ, Thirumalai D (1995) Modeling the role of disulfide bonds in protein folding: entropic barriers and pathways. Proteins 22(1):27–40PubMedGoogle Scholar
  20. Chan HS, Zhang Z et al (2011) Cooperativity, local-non-local coupling, and nonnative interactions: principles of protein folding from coarse-grained models. Annu Rev Phys Chem 62:301–326Google Scholar
  21. Chikenji G, Fujitsuka Y et al (2006) Shaping up the protein folding funnel by local interaction: lesson from a structure prediction study. Proc Natl Acad Sci USA 103(9):3141–3146PubMedGoogle Scholar
  22. Chintapalli SV, Yew BK et al (2010) Closed loop folding units from structural alignments: experimental foldons revisited. J Comput Chem 31(15):2689–2701PubMedGoogle Scholar
  23. Dadlez M (1999) Folding initiation sites and protein folding. Acta Biochim Pol 46(3):487–508PubMedGoogle Scholar
  24. Daggett V, Fersht AR (2003) Is there a unifying mechanism for protein folding? Trends Biochem Sci 28(1):18–25PubMedGoogle Scholar
  25. Dill KA, Fiebig KM et al (1993) Cooperativity in protein-folding kinetics. Proc Natl Acad Sci USA 90(5):1942–1946PubMedGoogle Scholar
  26. Dill KA, Ozkan SB et al (2008) The protein folding problem. Annu Rev Biophys 37:289–316PubMedGoogle Scholar
  27. Dokholyan NV, Buldyrev SV et al (2000) Identifying the protein folding nucleus using molecular dynamics. J Mol Biol 296(5):1183–1188PubMedGoogle Scholar
  28. Englander SW, Mayne L et al (2007) Protein folding and misfolding: mechanism and principles. Q Rev Biophys 40(4):287–326PubMedGoogle Scholar
  29. Felitsky DJ, Lietzow MA et al (2008) Modeling transient collapsed states of an unfolded protein to provide insights into early folding events. Proc Natl Acad Sci USA 105(17):6278–6283PubMedGoogle Scholar
  30. Fersht AR (1995) Optimization of rates of protein folding: the nucleation-condensation mechanism and its implications. Proc Natl Acad Sci USA 92(24):10869–10873PubMedGoogle Scholar
  31. Fersht AR (1997) Nucleation mechanisms in protein folding. Curr Opin Struct Biol 7(1):3–9PubMedGoogle Scholar
  32. Fersht AR (2000) Transition-state structure as a unifying basis in protein-folding mechanisms: contact order, chain topology, stability, and the extended nucleus mechanism. Proc Natl Acad Sci USA 97(4):1525–1529PubMedGoogle Scholar
  33. Fersht AR, Daggett V (2002) Protein folding and unfolding at atomic resolution. Cell 108(4):573–582PubMedGoogle Scholar
  34. Finkelstein AV, Shakhnovich EI (1989) Theory of cooperative transitions in protein molecules. II. Phase diagram for a protein molecule in solution. Biopolymers 28(10):1681–1694PubMedGoogle Scholar
  35. Fulton KF, Main ER et al (1999) Mapping the interactions present in the transition state for unfolding/folding of FKBP12. J Mol Biol 291(2):445–461PubMedGoogle Scholar
  36. Geierhaas CD, Paci E et al (2004) Comparison of the transition states for folding of two Ig-like proteins from different superfamilies. J Mol Biol 343(4):1111–1123PubMedGoogle Scholar
  37. Go N, Taketomi H (1978) Respective roles of short- and long-range interactions in protein folding. Proc Natl Acad Sci USA 75(2):559–563PubMedGoogle Scholar
  38. Goldstein RA, Luthey-Schulten ZA et al (1992) Optimal protein-folding codes from spin-glass theory. Proc Natl Acad Sci USA 89(11):4918–4922PubMedGoogle Scholar
  39. Gromiha MM, Selvaraj S (2001) Comparison between long-range interactions and contact order in determining the folding rate of two-state proteins: application of long-range order to folding rate prediction. J Mol Biol 310(1):27–32PubMedGoogle Scholar
  40. Gulotta M, Gilmanshin R et al (2001) Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape. Biochemistry 40(17):5137–5143PubMedGoogle Scholar
  41. Haas E (2005) The study of protein folding and dynamics by determination of intramolecular distance distributions and their fluctuations using ensemble and single-molecule FRET measurements. Chemphyschem 6(5):858–870PubMedGoogle Scholar
  42. Harrison SC, Durbin R (1985) Is there a single pathway for the folding of a polypeptide chain? Proc Natl Acad Sci USA 82(12):4028–4030PubMedGoogle Scholar
  43. Haspel N, Tsai CJ et al (2003a) Hierarchical protein folding pathways: a computational study of protein fragments. Proteins 51(2):203–215PubMedGoogle Scholar
  44. Haspel N, Tsai CJ et al (2003b) Reducing the computational complexity of protein folding via fragment folding and assembly. Protein Sci 12(6):1177–1187PubMedGoogle Scholar
  45. Hoang L, Maity H et al (2003) Folding units govern the cytochrome c alkaline transition. J Mol Biol 331(1):37–43PubMedGoogle Scholar
  46. Hubner IA, Edmonds KA et al (2005) Nucleation and the transition state of the SH3 domain. J Mol Biol 349(2):424–434PubMedGoogle Scholar
  47. Hubner IA, Oliveberg M et al (2004a) Simulation, experiment, and evolution: understanding nucleation in protein S6 folding. Proc Natl Acad Sci USA 101(22):8354–8359PubMedGoogle Scholar
  48. Hubner IA, Shimada J et al (2004b) Commitment and nucleation in the protein G transition state. J Mol Biol 336(3):745–761PubMedGoogle Scholar
  49. Ionescu RM, Matthews CR (1999) Folding under the influence. Nat Struct Biol 6(4):304–307PubMedGoogle Scholar
  50. Ittah V, Haas E (1995) Non-local interactions stabilize long range loops in the initial folding intermediates of reduced bovine pancreatic trypsin inhibitor. Biochemistry 34(13):4493–4506PubMedGoogle Scholar
  51. Ivankov DN, Garbuzynskiy SO et al (2003) Contact order revisited: influence of protein size on the folding rate. Protein Sci 12(9):2057–2062PubMedGoogle Scholar
  52. Jacob MH, Amir D et al (2005) Predicting reactivities of protein surface cysteines as part of a strategy for selective multiple labeling. Biochemistry 44(42):13664–13672PubMedGoogle Scholar
  53. Juraszek J, Bolhuis PG (2006) Sampling the multiple folding mechanisms of Trp-cage in explicit solvent. Proc Natl Acad Sci USA 103(43):15859–15864PubMedGoogle Scholar
  54. Karplus M, Weaver DL (1976) Protein-folding dynamics. Nature 260(5550):404–406PubMedGoogle Scholar
  55. Kato S, Kamikubo H et al (2010) non-local interactions are responsible for tertiary structure formation in staphylococcal nuclease. Biophys J 98(4):678–686PubMedGoogle Scholar
  56. Kihara D (2005) The effect of long-range interactions on the secondary structure formation of proteins. Protein Sci 14(8):1955–1963PubMedGoogle Scholar
  57. Kim PS, Baldwin RL (1982) Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Annu Rev Biochem 51:459–489PubMedGoogle Scholar
  58. Kimura T, Lee JC et al (2007) Site-specific collapse dynamics guide the formation of the cytochrome c’ four-helix bundle. Proc Natl Acad Sci USA 104(1):117–122PubMedGoogle Scholar
  59. Krantz BA, Dothager RS et al (2004) Discerning the structure and energy of multiple transition states in protein folding using psi-analysis. J Mol Biol 337(2):463–475PubMedGoogle Scholar
  60. Krantz BA, Mayne L et al (2002) Fast and slow intermediate accumulation and the initial barrier mechanism in protein folding. J Mol Biol 324(2):359–371PubMedGoogle Scholar
  61. Krieger F, Fierz B et al (2003) Dynamics of unfolded polypeptide chains as model for the earliest steps in protein folding. J Mol Biol 332(1):265–274PubMedGoogle Scholar
  62. Lapidus LJ, Eaton WA et al (2000) Measuring the rate of intramolecular contact formation in polypeptides. Proc Natl Acad Sci USA 97(13):7220–7225PubMedGoogle Scholar
  63. Lapidus LJ, Yao S et al (2007) Protein hydrophobic collapse and early folding steps observed in a microfluidic mixer. Biophys J 93(1):218–224PubMedGoogle Scholar
  64. Lappalainen I, Hurley MG et al (2008) Plasticity within the obligatory folding nucleus of an immunoglobulin-like domain. J Mol Biol 375(2):547–559PubMedGoogle Scholar
  65. Lee J, Liwo A et al (1999) Energy-based de novo protein folding by conformational space annealing and an off-lattice united-residue force field: application to the 10–55 fragment of staphylococcal protein A and to apo calbindin D9K. Proc Natl Acad Sci USA 96(5):2025–2030PubMedGoogle Scholar
  66. Levitt M (1992) Accurate modeling of protein conformation by automatic segment matching. J Mol Biol 226(2):507–533PubMedGoogle Scholar
  67. Lindorff-Larsen K, Rogen P et al (2005) Protein folding and the organization of the protein topology universe. Trends Biochem Sci 30(1):13–19PubMedGoogle Scholar
  68. Lindorff-Larsen K, Vendruscolo M et al (2004) Transition states for protein folding have native topologies despite high structural variability. Nat Struct Mol Biol 11(5):443–449PubMedGoogle Scholar
  69. Lonquety M, Chomilier J et al (2009) Prediction of stability upon point mutation in the context of the folding nucleus. OMICS 14(2):151–156Google Scholar
  70. Meisner WK, Sosnick TR (2004) Fast folding of a helical protein initiated by the collision of unstructured chains. Proc Natl Acad Sci USA 101(37):13478–13482PubMedGoogle Scholar
  71. Mirny L, Shakhnovich E (2001) Protein folding theory: from lattice to all-atom models. Annu Rev Biophys Biomol Struct 30:361–396PubMedGoogle Scholar
  72. Moult J, Unger R (1991) An analysis of protein folding pathways. Biochemistry 30(16):3816–3824PubMedGoogle Scholar
  73. Muller CW, Schulz GE (1988) Structure of the complex of adenylate kinase from Escherichia coli with the inhibitor P1, P5-di(adenosine-5′-)pentaphosphate. J Mol Biol 202(4):909–912PubMedGoogle Scholar
  74. Muller CW, Schulz GE (1992) Structure of the complex between adenylate kinase from Escherichia coli and the inhibitor Ap5A refined at 1.9 A resolution. A model for a catalytic transition state. J Mol Biol 224(1):159–177PubMedGoogle Scholar
  75. Munson M, Anderson KS et al (1997) Speeding up protein folding: mutations that increase the rate at which Rop folds and unfolds by over four orders of magnitude. Fold Des 2(1):77–87PubMedGoogle Scholar
  76. Nishimura C, Lietzow MA et al (2005) Sequence determinants of a protein folding pathway. J Mol Biol 351(2):383–392PubMedGoogle Scholar
  77. Noda L, Schulz GE et al (1975) Crystalline adenylate kinase from carp muscle. Eur J Biochem 51(1):229–235PubMedGoogle Scholar
  78. Noivirt-Brik O, Hazan G et al (2013) Non local residue-residue contacts in proteins are more conserved than local ones. Bioinformatics 29(3):331–337Google Scholar
  79. Northey JG, Di Nardo AA et al (2002) Hydrophobic core packing in the SH3 domain folding transition state. Nat Struct Biol 9(2):126–130PubMedGoogle Scholar
  80. O’Neill JC Jr, Robert Matthews C (2000) Localized, stereochemically sensitive hydrophobic packing in an early folding intermediate of dihydrofolate reductase from Escherichia coli. J Mol Biol 295(4):737–744PubMedGoogle Scholar
  81. Orevi T, Ben Ishay E et al (2009) Early closure of a long loop in the refolding of adenylate kinase: a possible key role of non-local interactions in the initial folding steps. J Mol Biol 385(4):1230–1242PubMedGoogle Scholar
  82. Ozkan SB, Wu GA et al (2007) Protein folding by zipping and assembly. Proc Natl Acad Sci USA 104(29):11987–11992PubMedGoogle Scholar
  83. Paci E, Clarke J et al (2003) Self-consistent determination of the transition state for protein folding: application to a fibronectin type III domain. Proc Natl Acad Sci USA 100(2):394–399PubMedGoogle Scholar
  84. Papandreou N, Berezovsky IN et al (2004) Universal positions in globular proteins. Eur J Biochem 271(23–24):4762–4768PubMedGoogle Scholar
  85. Plaxco KW, Simons KT et al (1998) Contact order, transition state placement and the refolding rates of single domain proteins. J Mol Biol 277(4):985–994PubMedGoogle Scholar
  86. Prudhomme N, Chomilier J (2009) Prediction of the protein folding core: application to the immunoglobulin fold. Biochimie 91(11–12):1465–1474PubMedGoogle Scholar
  87. Ptitsyn OB (1973) Stages in the mechanism of self-organization of protein molecules. Dokl Akad Nauk SSSR 210(5):1213–1215PubMedGoogle Scholar
  88. Ratner V, Amir D et al (2005) Fast collapse but slow formation of secondary structure elements in the refolding transition of E. coli adenylate kinase. J Mol Biol 352(3):683–699PubMedGoogle Scholar
  89. Ratner V, Sinev M et al (2000) Determination of intramolecular distance distribution during protein folding on the millisecond timescale. J Mol Biol 299(5):1363–1371PubMedGoogle Scholar
  90. Rooman MJ, Kocher JP et al (1992) Extracting information on folding from the amino acid sequence: accurate predictions for protein regions with preferred conformation in the absence of tertiary interactions. Biochemistry 31(42):10226–10238PubMedGoogle Scholar
  91. Sali A, Shakhnovich E et al (1994) How does a protein fold? Nature 369(6477):248–251PubMedGoogle Scholar
  92. Samatova EN, Katina NS et al (2009) How strong are side chain interactions in the folding intermediate? Protein Sci 18(10):2152–2159PubMedGoogle Scholar
  93. Scalley-Kim M, Minard P et al (2003) Low free energy cost of very long loop insertions in proteins. Protein Sci 12(2):197–206PubMedGoogle Scholar
  94. Schulz GE, Muller CW et al (1990) Induced-fit movements in adenylate kinases. J Mol Biol 213(4):627–630PubMedGoogle Scholar
  95. Shakhnovich EI (1994) Proteins with selected sequences fold into unique native conformation. Phys Rev Lett 72(24):3907–3910PubMedGoogle Scholar
  96. Shakhnovich EI, Gutin AM (1993a) Engineering of stable and fast-folding sequences of model proteins. Proc Natl Acad Sci USA 90(15):7195–7199PubMedGoogle Scholar
  97. Shakhnovich EI, Gutin AM (1993b) A new approach to the design of stable proteins. Protein Eng 6(8):793–800PubMedGoogle Scholar
  98. Shell MS, Ozkan SB et al (2009) Blind test of physics-based prediction of protein structures. Biophys J 96(3):917–924PubMedGoogle Scholar
  99. Shortle D, Meeker AK (1989) Residual structure in large fragments of staphylococcal nuclease: effects of amino acid substitutions. Biochemistry 28(3):936–944PubMedGoogle Scholar
  100. Simons KT, Kooperberg C et al (1997) Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions. J Mol Biol 268(1):209–225PubMedGoogle Scholar
  101. Sinha KK, Udgaonkar JB (2007) Dissecting the non-specific and specific components of the initial folding reaction of barstar by multi-site FRET measurements. J Mol Biol 370(2):385–405PubMedGoogle Scholar
  102. Sosnick TR, Dothager RS et al (2004) Differences in the folding transition state of ubiquitin indicated by phi and psi analyses. Proc Natl Acad Sci USA 101(50):17377–17382PubMedGoogle Scholar
  103. Sosnick TR, Mayne L et al (1994) The barriers in protein folding. Nat Struct Biol 1(3):149–156PubMedGoogle Scholar
  104. Steward A, McDowell GS et al (2009) Topology is the principal determinant in the folding of a complex all-alpha Greek key death domain from human FADD. J Mol Biol 389(2):425–437PubMedGoogle Scholar
  105. Taketomi H, Ueda Y et al (1975) Studies on protein folding, unfolding and fluctuations by computer simulation. I. The effect of specific amino acid sequence represented by specific inter-unit interactions. Int J Pept Protein Res 7(6):445–459PubMedGoogle Scholar
  106. Teufel DP, Johnson CM et al (2011) Backbone-driven collapse in unfolded protein chains. J Mol Biol 409(2):250–262PubMedGoogle Scholar
  107. Tsong TY, Hu CK et al (2008) Hydrophobic condensation and modular assembly model of protein folding. Biosystems 93(1–2):78–89PubMedGoogle Scholar
  108. Unger R, Moult J (1996) Local interactions dominate folding in a simple protein model. J Mol Biol 259(5):988–994PubMedGoogle Scholar
  109. Wang L, Rivera EV et al (2005) Loop entropy and cytochrome c stability. J Mol Biol 353(3):719–729PubMedGoogle Scholar
  110. Weikl TR (2008) Loop-closure principles in protein folding. Arch Biochem Biophys 469(1):67–75PubMedGoogle Scholar
  111. Weikl TR, Dill KA (2003) Folding rates and low-entropy-loss routes of two-state proteins. J Mol Biol 329(3):585–598PubMedGoogle Scholar
  112. Wetlaufer DB (1973) Nucleation, rapid folding, and globular intrachain regions in proteins. Proc Natl Acad Sci USA 70(3):697–701PubMedGoogle Scholar
  113. Wright PE, Dyson HJ et al (1988) Conformation of peptide fragments of proteins in aqueous solution: implications for initiation of protein folding. Biochemistry 27(19):7167–7175PubMedGoogle Scholar
  114. Wu Y, Kondrashkina E et al (2008) Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein. Proc Natl Acad Sci USA 105(36):13367–13372PubMedGoogle Scholar
  115. Zhang Z, Chan HS (2013) Transition paths, diffusive processes, and preequilibria of protein folding. Proc Natl Acad Sci USA 109(51):20919–20924Google Scholar

Copyright information

© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Tomer Orevi
    • 1
  • Gil Rahamim
    • 1
  • Gershon Hazan
    • 1
  • Dan Amir
    • 1
  • Elisha Haas
    • 1
  1. 1.The Goodman Faculty of Life SciencesBar Ilan UniversityRamat GanIsrael

Personalised recommendations