Biophysical Reviews

, Volume 5, Issue 2, pp 137–145 | Cite as

Effects of macromolecular crowding agents on protein folding in vitro and in silico

  • Alexander Christiansen
  • Qian Wang
  • Margaret S. Cheung
  • Pernilla Wittung-StafshedeEmail author


Proteins fold and function inside cells which are environments very different from that of dilute buffer solutions most often used in traditional experiments. The crowded milieu results in excluded-volume effects, increased bulk viscosity and amplified chances for inter-molecular interactions. These environmental factors have not been accounted for in most mechanistic studies of protein folding executed during the last decades. The question thus arises as to how these effects—present when polypeptides normally fold in vivo—modulate protein biophysics. To address excluded volume effects, we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with proteins, in combination with strategically selected proteins and a range of equilibrium and time-resolved biophysical (spectroscopic and computational) methods. In this review, we describe key observations on macromolecular crowding effects on protein stability, folding and structure drawn from combined in vitro and in silico studies. As expected based on Minton’s early predictions, many proteins (apoflavodoxin, VlsE, cytochrome c, and S16) became more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, unexpectedly, for apoflavodoxin and VlsE, the folded states changed both secondary structure content and, for VlsE, overall shape in the presence of macromolecular crowding. For apoflavodoxin and cytochrome c, which have complex kinetic folding mechanisms, excluded volume effects made the folding energy landscapes smoother (i.e., less misfolding and/or kinetic heterogeneity) than in buffer.


Protein folding Macromolecular crowding Spectroscopy Protein stability Excluded volume Coarse-grained simulation 



We would like to thank Allen Minton for his continuous (careful and sometimes critical) feedback on our work on crowding. Minton’s pioneering theoretical contributions to this field has served as the basis for many of our studies and they continue to stimulate new experiments. We thank Eefei Chen, University of California, Santa Cruz for preparing Fig. 3 and Jörgen Åden, Umeå University for preparing Fig. 1; and Magnus Wolf-Watz, Umeå University for helpful comments on the text.

Conflict of interest statement

The authors declare that they have no conflict of interest.


  1. Aguilar X, Weise FC et al (2011) Macromolecular crowding extended to a heptameric system: the Co-chaperonin protein 10. Biochemistry 50(14):3034–3044PubMedCrossRefGoogle Scholar
  2. Ai X, Zhou Z, Choy WY (2006) 15N NMR spin relaxation dispersion study of the molecular crowding effects on protein folding under native conditions. J Am Chem Soc 128:3916–3917Google Scholar
  3. Batra J, Xu K et al (2009) Nonadditive effects of mixed crowding on protein stability. Proteins 77(1):133–138PubMedCrossRefGoogle Scholar
  4. Benton LA, Smith AE et al (2012) Unexpected Effects of Macromolecular Crowding on Protein Stability. Biochemistry (in press)Google Scholar
  5. Bertini I, Gary HB et al (1994) Bioinorganic chemistry. University Science, Mill ValleyGoogle Scholar
  6. Bohrer MP, Patterson GD, Carroll PJ (1984) Hindered diffusion of dextran and Ficoll in microporous membranes. Macromolecules 17:1170–1173CrossRefGoogle Scholar
  7. Bryngelson JD, Onuchic JN et al (1995) Funnels, pathways, and the energy landscape of protein folding: a synthesis. Proteins 21(3):167–195PubMedCrossRefGoogle Scholar
  8. Burston SG, Clarke AR (1995) Molecular chaperones: physical and mechanistic properties. Essays Biochem 29:125–136PubMedGoogle Scholar
  9. Chen E, Christiansen A et al (2012) The effects of macromolecular crowding on burst phase kinetics of cytochrome c folding. Biochemistry 51(49):9836–9845Google Scholar
  10. Cheung MS, Klimov D et al (2005) Molecular crowding enhances native state stability and refolding rates of globular proteins. Proc Natl Acad Sci USA 102(13):4753–4758PubMedCrossRefGoogle Scholar
  11. Christiansen A, Wang Q et al (2010) Factors defining effects of macromolecular crowding on protein stability: an in vitro/in silico case study using cytochrome c. Biochemistry 49(31):6519–6530PubMedCrossRefGoogle Scholar
  12. Dedmon MM, Patel CN et al (2002) FlgM gains structure in living cells. Proc Natl Acad Sci USA 99(20):12681–12684PubMedCrossRefGoogle Scholar
  13. Dhar A, Samiotakis A et al (2010) Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding. Proc Natl Acad Sci USA 107(41):17586–17591PubMedCrossRefGoogle Scholar
  14. Dhar A, Girdhar K et al (2011) Protein stability and folding kinetics in the nucleus and endoplasmic reticulum of eucaryotic cells. Biophys J 101(2):421–430PubMedCrossRefGoogle Scholar
  15. Engel R, Westphal AH et al (2008) Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin folding. J Biol Chem 283(41):27383–27394PubMedCrossRefGoogle Scholar
  16. Flaugh SL, Lumb KJ (2001) Effects of macromolecular crowding on the intrinsically disordered proteins c-Fos and p27(Kip1). Biomacromolecules 2(2):538–540PubMedCrossRefGoogle Scholar
  17. Frydman J (2001) Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu Rev Biochem 70:603–647PubMedCrossRefGoogle Scholar
  18. Homouz D, Perham M et al (2008) Crowded, cell-like environment induces shape changes in aspherical protein. Proc Natl Acad Sci USA 105(33):11754–11759PubMedCrossRefGoogle Scholar
  19. Homouz D, Sanabria H et al (2009a) Modulation of calmodulin plasticity by the effect of macromolecular crowding. J Mol Biol 391(5):933–943PubMedCrossRefGoogle Scholar
  20. Homouz D, Stagg L et al (2009b) Macromolecular crowding modulates folding mechanism of alpha/beta protein apoflavodoxin. Biophys J 96(2):671–680PubMedCrossRefGoogle Scholar
  21. Hong J, Gierasch LM (2010) Macromolecular crowding remodels the energy landscape of a protein by favoring a more compact unfolded state. J Am Chem Soc 132(30):10445–10452PubMedCrossRefGoogle Scholar
  22. Johansen D, Jeffries CM et al (2011) Effects of macromolecular crowding on an intrinsically disordered protein characterized by small-angle neutron scattering with contrast matching. Biophys J 100(4):1120–1128PubMedCrossRefGoogle Scholar
  23. Khramtsov VV, Marsh D et al (1992) The application of pH-sensitive spin labels to studies of surface potential and polarity of phospholipid membranes and proteins. Biochim Biophys Acta 1104(2):317–324PubMedCrossRefGoogle Scholar
  24. Kliger DS, Chen E et al (2012) Nanosecond time-resolved natural and magnetic chiroptical spectroscopies. Wiley VCH, New YorkGoogle Scholar
  25. Kuhnert DC, Gildenhuys S, Dirr H (2008) Effect of macromolecular crowding on the stability of monomeric glutaredoxin 2 and dimeric glutathione transferase A1-1. S Afr J Sci 104:76–80Google Scholar
  26. Kulothungan SR, Das M, Johnson M, Ganesh C, Varadarajan R (2009) Effect of crowding agents, signal peptide, and chaperone SecB on the folding and aggregation of E. coli maltose binding protein.Langmuir 25:6637–6648Google Scholar
  27. Laurent TC, Ogston AG (1963) The interaction between polysaccharides and other macromolecules. 4. The osmotic pressure of mixtures of serum albumin and hyaluronic acid. Biochem J 89:249–253PubMedGoogle Scholar
  28. Lavalette D, Tetreau C et al (1999) Microscopic viscosity and rotational diffusion of proteins in a macromolecular environment. Biophys J 76(5):2744–2751PubMedCrossRefGoogle Scholar
  29. Lavalette D, Hink MA et al (2006) Proteins as micro viscosimeters: Brownian motion revisited. Eur Biophys J 35(6):517–522PubMedCrossRefGoogle Scholar
  30. Le Coeur C, Teixeira J et al (2010) Compression of random coils due to macromolecular crowding: scaling effects. Phys Rev E 81(6 Pt 1):061914CrossRefGoogle Scholar
  31. Lewis JW, Tilton RF et al (1985) New technique for measuring circular dichroism changes on a nanosecond time scale—application to (carbonmonoxy)myoglobin and (carbonmonoxy)hemoglobin. J Phys Chem 89(2):289–294CrossRefGoogle Scholar
  32. Li C, Pielak GJ (2009) Using NMR to distinguish viscosity effects from nonspecific protein binding under crowded conditions. J Am Chem Soc 131(4):1368–1369PubMedCrossRefGoogle Scholar
  33. Lutsenko S, LeShane ES et al (2007) Biochemical basis of regulation of human copper-transporting ATPases. Arch Biochem Biophys 463(2):134–148PubMedCrossRefGoogle Scholar
  34. Matouschek A, Kellis JT Jr et al (1989) Mapping the transition state and pathway of protein folding by protein engineering [see comments]. Nature 340(6229):122–126PubMedCrossRefGoogle Scholar
  35. McGuffee SR, Elcock AH (2010) Diffusion, crowding & protein stability in a dynamic molecular model of the bacterial cytoplasm. PLoS Comput Biol 6(3):e1000694PubMedCrossRefGoogle Scholar
  36. Mikaelsson T, Åden J et al (2013) Direct observation of protein unfolded state compaction in the presence of macromolecular crowding. Biophys J 104:694–704Google Scholar
  37. Miklos AC, Sarkar M et al (2011) Protein crowding tunes protein stability. J Am Chem Soc 133(18):7116–7120PubMedCrossRefGoogle Scholar
  38. Millett IS, Doniach S et al (2002) Toward a taxonomy of the denatured state: small angle scattering studies of unfolded proteins. Adv Protein Chem 62:241–262PubMedCrossRefGoogle Scholar
  39. Minton AP (1981) Excluded volume as a determinant of macromolecular structure and reactivity. Biopolymers 20:2093–2120CrossRefGoogle Scholar
  40. Minton AP (2000) Effect of a concentrated “inert” macromolecular cosolute on the stability of a globular protein with respect to denaturation by heat and by chaotropes: a statistical-thermodynamic model. Biophys J 78(1):101–109PubMedCrossRefGoogle Scholar
  41. Minton AP (2005) Models for excluded volume interaction between an unfolded protein and rigid macromolecular cosolutes: macromolecular crowding and protein stability revisited. Biophys J 88(2):971–985PubMedCrossRefGoogle Scholar
  42. Mukherjee S, Waegele MM et al (2009) Effect of macromolecular crowding on protein folding dynamics at the secondary structure level. J Mol Biol 393(1):227–236PubMedCrossRefGoogle Scholar
  43. Neuweiler H, Lollmann M et al (2007) Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy. J Mol Biol 365(3):856–869PubMedCrossRefGoogle Scholar
  44. Perham M, Stagg L et al (2007) Macromolecular crowding increases structural content of folded proteins. FEBS Lett 581(26):5065–5069PubMedCrossRefGoogle Scholar
  45. Phillip Y, Kiss V et al (2012) Protein-binding dynamics imaged in a living cell. Proc Natl Acad Sci USA 109(5):1461–1466PubMedCrossRefGoogle Scholar
  46. Pozdnyakova I, Wittung-Stafshede P (2010) Non-linear effects of macromolecular crowding on enzymatic activity of multi-copper oxidase. Biochim Biophys Acta 1804(4):740–744PubMedCrossRefGoogle Scholar
  47. Qu Y, Bolen DW (2002) Efficacy of macromolecular crowding in forcing proteins to fold. Biophys Chem 101–102:155–165PubMedCrossRefGoogle Scholar
  48. Rivas G, Ferrone F et al (2004) Life in a crowded world. EMBO Rep 5(1):23–27PubMedCrossRefGoogle Scholar
  49. Samiotakis A, Wittung-Stafshede P et al (2009) Folding, stability and shape of proteins in crowded environments: experimental and computational approaches. Int J Mol Sci 10(2):572–588PubMedCrossRefGoogle Scholar
  50. Sasahara K, McPhie P et al (2003) Effect of dextran on protein stability and conformation attributed to macromolecular crowding. J Mol Biol 326(4):1227–1237PubMedCrossRefGoogle Scholar
  51. Sasaki,Y, Miyoshi D, Sugimoto N (2007) Regulation of DNA nucleases bymolecular crowding. Nucleic Acids Res 35:4086–4093Google Scholar
  52. Schlesinger AP, Wang Y et al (2011) Macromolecular crowding fails to fold a globular protein in cells. J Am Chem Soc 133(21):8082–8085PubMedCrossRefGoogle Scholar
  53. Shaw MR, Thirumalai D (1991) Free polymer in a colloidal solution. Phys Rev A 44(8):R4797–R4800PubMedCrossRefGoogle Scholar
  54. Spencer DS,Xua K,Logan TM,Zhou H-X (2005) Effects of pH, salt, and macromolecular crowding on the stability of FK506-binding protein: an integrated experimental and theoretical study. J Mol Biol 351:219–232Google Scholar
  55. Stagg L, Zhang SQ et al (2007) Molecular crowding enhances native structure and stability of alpha/beta protein flavodoxin. Proc Natl Acad Sci USA 104(48):18976–18981PubMedCrossRefGoogle Scholar
  56. Stagg L, Christiansen A et al (2010a) Macromolecular crowding tunes folding landscape of parallel alpha/beta protein, apoflavodoxin. J Am Chem Soc 133(4):646–648PubMedCrossRefGoogle Scholar
  57. Stagg L, Samiotakis A et al (2010b) Residue-specific analysis of frustration in the folding landscape of repeat beta/alpha protein apoflavodoxin. J Mol Biol 396(1):75–89PubMedCrossRefGoogle Scholar
  58. Tellam RL, Sculley MJ, Nichol LW, Wills PR (1983)The influence of poly(ethylene glycol) 6000 on the properties of skeletal-muscle actin.Biochem J 213:651–659Google Scholar
  59. Tokuriki N, Kinjo M et al (2004) Protein folding by the effects of macromolecular crowding. Protein Sci 13(1):125–133PubMedCrossRefGoogle Scholar
  60. Venturoli D, Rippe B (2005) Ficoll and dextran vs. globular proteins as probes for testing glomerular permselectivity: effects of molecular size, shape, charge, and deformability. Am J Physiol Renal Physiol 288(4):F605–F613PubMedCrossRefGoogle Scholar
  61. Vopel T, Makhatadze GI (2012) Enzyme activity in the crowded milieu. PLoS One 7(6):e39418PubMedCrossRefGoogle Scholar
  62. Wang Y, Li C et al (2010) Effects of proteins on protein diffusion. J Am Chem Soc 132(27):9392–9397PubMedCrossRefGoogle Scholar
  63. Wang Q, Zhuravleva A et al (2011) Exploring weak, transient protein-protein interactions in crowded in vivo environments by in-cell nuclear magnetic resonance spectroscopy. Biochemistry 50(43):9225–9236PubMedCrossRefGoogle Scholar
  64. Wittung-Stafshede P (2011) Protein folding inside the cell. Biophys J 101(2):265–266PubMedCrossRefGoogle Scholar
  65. Zhou HX (2004) Loops, linkages, rings, catenanes, cages, and crowders: entropy based strategies for stabilizing proteins. Acc Chem Res 37:123–130PubMedCrossRefGoogle Scholar
  66. Zhou HX, Rivas G et al (2008) Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu Rev Biophys 37:375–397PubMedCrossRefGoogle Scholar
  67. Zimmerman SB, Trach SO (1991) Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J Mol Biol 222(3):599–620PubMedCrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Alexander Christiansen
    • 1
  • Qian Wang
    • 2
  • Margaret S. Cheung
    • 2
  • Pernilla Wittung-Stafshede
    • 1
    Email author
  1. 1.Department of ChemistryUmeå UniversityUmeåSweden
  2. 2.Department of PhysicsUniversity of HoustonHoustonUSA

Personalised recommendations