Biochemical Reactions in the Crowded and Confined Physiological Environment: Physical Chemistry Meets Synthetic Biology

  • Allen P. MintonEmail author
  • Germán Rivas


Proteins and nucleic acids constitute at least 20–30% of the total mass (and volume) of all living organisms without exception. Although local composition may vary widely with location within a given cell and between cells, it is evident that much of the chemistry of life – as opposed to laboratory biochemistry – takes place within media containing a substantial volume fraction of macromolecules. These media are termed “crowded” or “volume-occupied”, rather than “concentrated”, as no single macromolecular species need be concentrated. Moreover, many biological compartments do not consist of a continuous fluid phase, but rather a series of small interstitial elements of fluid, or “pores”, bounded by membranes or other relatively immobile structural elements such as cytoskeletal filaments. Such interstitial volume elements might be likened to the holes in a sponge, except that the characteristic sizes of the “holes” are of the order of tens of nanometers. The soluble macromolecules within these pores are termed “confined” to reflect the discontinuous nature of the fluid phase and the small dimensions of the pores.


Synthetic Biology Standard Free Energy Relative Free Energy Exclude Volume Effect Macromolecular Crowding 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Research conducted in A.P.M.’s laboratory is supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. The author thanks Peter McPhie (NIH) for reviewing drafts of this Commentary.

Research in GR lab is funded by the Spanish Ministry of Science and Innovation (grant BIO2008-04478-C03-03), the Madrid Government (COMBACT_CM), and the EU (HEALTH-F3-2009-223431).

We are grateful to Company of Biologists Ltd., which allowed the reproduction of the full article (Minton 2006).


  1. Arbuzova A, Murray D, McLaughlin S (1998) MARCKS, membranes, and calmodulin: kinetics of their interaction. Biochim Biophys Acta 1376:369–379PubMedGoogle Scholar
  2. Blanco R, Arai A, Grinberg N, Yarmush DM, Karger BL (1989) Role of association on protein adsorption isotherms. Beta-lactoglobulin A adsorbed on a weakly hydrophobic surface. J Chromatogr 482:1–12CrossRefPubMedGoogle Scholar
  3. Bloemendal H, de Jong W, Jaenicke R, Lubsen NH, Slingsby C, Tardieu A (2004) Ageing and vision: structure, stability and function of lens crystallins. Prog Biophys Mol Biol 86:407–485CrossRefPubMedGoogle Scholar
  4. Bolis D, Politou AS, Kelly G, Pastore A, Temussi PA (2004) Protein stability in nanocages: a novel approach for influencing protein stability by molecular confinement. J Mol Biol 336:203–212CrossRefPubMedGoogle Scholar
  5. Bookchin RM, Balasz T, Wang Z, Josephs R, Lew VL (1999) Polymer structure and solubility of deoxyhemoglobin S in the presence of high concentrations of volume-excluding 70-kDa dextran. Effects of non-S hemoglobins and inhibitors. J Biol Chem 274:6689–6697CrossRefPubMedGoogle Scholar
  6. Chatelier RC, Minton AP (1996) Adsorption of globular proteins on locally planar surfaces: models for the effect of excluded surface area and aggregation of adsorbed protein on adsorption equilibria. Biophys J 71:2367–2374CrossRefPubMedGoogle Scholar
  7. Cheung MS, Thirumalai D (2006) Nanopore-protein interactions dramatically alter stability and yield of the native state in restricted spaces. J Mol Biol 357:632643CrossRefGoogle Scholar
  8. Cheung MS, Klimov D, Thirumalai D (2005) Molecular crowding enhances native state stability and refolding rates of globular proteins. Proc Natl Acad Sci USA 102:4753–4758CrossRefPubMedGoogle Scholar
  9. Colclasure GC, Parker JC (1992) Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells. J Gen Physiol 100:1–10CrossRefPubMedGoogle Scholar
  10. Cutsforth G, Whitaker R, Hermans J, Lentz B (1989) A new model to describe extrinsic protein binding to phospholipid membranes of varying composition: application to human coagulation proteins. Biochemistry 28:7453–7461CrossRefPubMedGoogle Scholar
  11. Darst SA, Ribi HO, Pierce DW, Kornberg RD (1988) Two-dimensional crystals of E. coli RNA polymerase holoenzyme on positively charged lipid layers. J Mol Biol 203:269–273CrossRefPubMedGoogle Scholar
  12. del Alamo M, Rivas G, Mateu MG (2005) Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro. J Virol 79:14271–14281CrossRefPubMedGoogle Scholar
  13. Drenckhahn D, Pollard TD (1986) Elongation of actin filaments is a diffusion limited reaction at the barbed end and is accelerated by inert macromolecules. J Biol Chem 261:12754–12758PubMedGoogle Scholar
  14. Edwards RA, Huber RE (1992) Surface denaturation of proteins: the thermal inactivation of beta-galactosidase (Escherichia coli) on wall-liquid surfaces. Biochem Cell Biol 70:63–69CrossRefPubMedGoogle Scholar
  15. Eggers D, Valentine J (2001) Molecular confinement influences protein structure and enhances thermal protein stability. Protein Sci 10:250–261CrossRefPubMedGoogle Scholar
  16. Fernandez A, Berry RS (2003) Proteins with H-bond packing defects are highly interactive with lipid bilayers: implications for amyloidogenesis. Proc Natl Acad Sci USA 100:2391–2396CrossRefPubMedGoogle Scholar
  17. Ferrone F (2004) Polymerization and sickle cell disease: a molecular view. Microcirculation 11:115–128PubMedGoogle Scholar
  18. Fulton AB (1982) How crowded is the cytoplasm? Cell 30:345–347CrossRefPubMedGoogle Scholar
  19. Ghaemmaghami S, Oas TG (2001) Quantitative protein stability measurement in vivo. Nat Struct Biol 8:879–882CrossRefPubMedGoogle Scholar
  20. Giddings JC, Kucera E, Russell CP, Myers MN (1968) Statistical theory for the equilibrium distribution of rigid molecules in inert porous networks. Exclusion chromatography. J Phys Chem 72:4397–4408CrossRefGoogle Scholar
  21. Goodsell DS (1993) The machinery of life. Springer, New YorkGoogle Scholar
  22. Hall D, Minton AP (2002) Effects of inert volume-excluding macromolecules on protein fiber formation I. Equilibrium models. Biophys Chem 98:93–104CrossRefPubMedGoogle Scholar
  23. Hall D, Minton AP (2004) Effects of inert volume-excluding macromolecules on protein fiber formation II. Kinetic models for nucleated fiber growth. Biophys Chem 107:299–316CrossRefPubMedGoogle Scholar
  24. Hatters D, Minton AP, Howlett GJ (2002) Macromolecular crowding accelerates amyloid formation by human apolipoprotein C-II. J Biol Chem 277:7824–7830CrossRefPubMedGoogle Scholar
  25. Herzog W, Weber K (1978) Microtubule formation by pure brain tubulin in vitro. The influence of dextran and polyethylene glycol. Eur J Biochem 91:249–254CrossRefPubMedGoogle Scholar
  26. Ignatova Z, Gierasch LM (2004) Monitoring protein stability and aggregation in vivo by real time fluorescent labeling. Proc Natl Acad Sci USA 101:523–528CrossRefPubMedGoogle Scholar
  27. Jarvis TC, Ring DM, Daube SS, von Hippel PH (1990) “Macromolecular crowding”: thermodynamic consequences for protein-protein interactions within the T4 DNA replication complex. J Biol Chem 265:15160–15167PubMedGoogle Scholar
  28. Klimov DK, Newfield D, Thirumalai D (2002) Simulations of beta-hairpin folding confined to spherical pores using distributed computing. Proc Natl Acad Sci USA 99:8019–8024CrossRefPubMedGoogle Scholar
  29. Knull HR, Walsh JL (1992) Association of glycolytic enzymes with the cytoskeleton. Curr Top Cell Regul 33:15–30PubMedGoogle Scholar
  30. Knull H, Minton AP (1996) Structure within eukaryotic cytoplasm and its relationship to glycolytic metabolism. Cell Biochem Funct 14:237–248CrossRefPubMedGoogle Scholar
  31. Koo EH, Lansbury PT, Kelly JW (1999) Amyloid diseases: abnormal protein aggregation in neurodegeneration. Proc Natl Acad Sci USA 96:9989–9990CrossRefPubMedGoogle Scholar
  32. Kozer N, Schreiber G (2004) Effect of crowding on protein-protein association rates: fundamental differences between low and high mass crowding agents. J Mol Biol 336:763–774CrossRefPubMedGoogle Scholar
  33. Lakatos S, Minton AP (1991) Interactions between globular proteins and F-actin in isotonic saline solution. J Biol Chem 266:18707–18713PubMedGoogle Scholar
  34. Lang F, Busch GL, Ritter M, Völkl H, Waldegger S, Gulbins E, Häussinger D (1998) Functional significance of cell volume regulatory mechanisms. Physiol Rev 78:247–306PubMedGoogle Scholar
  35. Lebowitz JL, Helfand E, Praestgaard E (1965) Scaled particle theory of fluid mixtures. J Chem Phys 43:774–779CrossRefGoogle Scholar
  36. Lindner R, Ralston G (1995) Effects of dextran on the self-association of human spectrin. Biophys Chem 57:15–25CrossRefPubMedGoogle Scholar
  37. Lindner RA, Ralston GB (1997) Macromolecular crowding: effects on actin polymerization. Biophys Chem 66:57–66CrossRefPubMedGoogle Scholar
  38. Martin J (2002) Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding. Biochemistry 41:5050–5055CrossRefPubMedGoogle Scholar
  39. May A, Huehns ER (1975) The concentration dependence of the oxygen affinity of haemoglobin. S Br J Haematol 30:317–335CrossRefGoogle Scholar
  40. McNulty BC, Young GB, Pielak GJ (2006) Macromolecular crowding in the Escherichia coli periplasm maintains alpha-synuclein disorder. J Mol Biol 355:893–897CrossRefPubMedGoogle Scholar
  41. Minton AP (1976) Quantitative relations between oxygen saturation and aggregation of sickle-cell hemoglobin: analysis of oxygen binding data. In: Hercules JI, Cottam GL, Waterman MR, Schechter AN (eds) Proceedings of the symposium on molecular and cellular aspects of sickle cell disease, pp 257–273. U.S. Department of Health, Education and Welfare, Bethesda, MDGoogle Scholar
  42. Minton AP (1981) Excluded volume as a determinant of macromolecular structure and reactivity. Biopolymers 20:2093–2120CrossRefGoogle Scholar
  43. Minton AP (1983) The effect of volume occupancy upon the thermodynamic activity of proteins: some biochemical consequences. Mol Cell Biochem 55:119–140CrossRefPubMedGoogle Scholar
  44. Minton AP (1989) Holobiochemistry: an integrated approach to the understanding of biochemical mechanism that emerges from the study of proteins and protein associations in volume-occupied solutions. In: Srere P, Jones ME, Mathews C (eds) Structural and Organizational Aspects of Metabolic Regulation. Alan R. Liss, New York, pp 291–306Google Scholar
  45. Minton AP (1992) Confinement as a determinant of macromolecular structure and reactivity. Biophys J 63:1090–1100CrossRefPubMedGoogle Scholar
  46. Minton AP (1995) Confinement as a determinant of macromolecular structure and reactivity. II. Effects of weakly attractive interactions between confined macrosolutes and confining structures. Biophys J 68:1311–1322CrossRefPubMedGoogle Scholar
  47. Minton AP (1998) Molecular crowding: analysis of effects of high concentrations of inert cosolutes on biochemical equilibria and rates in terms of volume exclusion. Meth Enzymol 295:127–149CrossRefPubMedGoogle Scholar
  48. Minton AP (2000a) 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 78:101–109CrossRefPubMedGoogle Scholar
  49. Minton AP (2000b) Effects of excluded surface area and adsorbate clustering on surface adsorption isotherms I. Equilibrium models. Biophys Chem 86:239–247CrossRefPubMedGoogle Scholar
  50. Minton AP (2001a) The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. J Biol Chem 276:10577–10580CrossRefPubMedGoogle Scholar
  51. Minton AP (2001b) Effects of excluded surface area and adsorbate clustering on surface adsorption of proteins II. Kinetic models. Biophys J 80:1641–1648CrossRefPubMedGoogle Scholar
  52. Minton AP (2006) How can biochemical reactions within cells differ from those in test tubes? J Cell Sci 119(14):2863–2869CrossRefPubMedGoogle Scholar
  53. Minton AP, Wilf J (1981) Effect of macromolecular crowding upon the structure and function of an enzyme: glyceraldehyde-3-phosphate dehydrogenase. Biochemistry 20:4821–4826CrossRefPubMedGoogle Scholar
  54. Nichol L, Ogston A, Wills P (1981) Effect of inert polymers on protein self association. FEBS Lett 126:18–20CrossRefPubMedGoogle Scholar
  55. Nygren H, Stenberg M (1990) Surface-induced aggregation of ferritin: kinetics of adsorption to a hydrophobic surface. Biophys Chem 38:67–75CrossRefPubMedGoogle Scholar
  56. Parameswaran S, Barber BJ, Babbit RA, Dutta S (1995) Age-related changes in albumin-excluded volume fraction. Microvasc Res 50:373–380CrossRefPubMedGoogle Scholar
  57. Ramsden JJ, Bachmanova GI, Archakov AI (1994) Kinetic evidence for protein clustering at a surface. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics 50:5072–5076PubMedGoogle Scholar
  58. Rivas G, Fernandez JA, Minton AP (1999) Direct observation of the self association of dilute proteins in the presence of inert macromolecules at high concentration via tracer sedimentation equilibrium: theory, experiment, and biological significance. Biochemistry 38:9379–9388CrossRefPubMedGoogle Scholar
  59. Rivas G, Fernandez JA, Minton AP (2001) Direct observation of the enhancement of non-cooperative protein self-assembly by macromolecular crowding: indefinite linear self-association of bacterial cell division protein FtsZ. Proc Natl Acad Sci USA 98:3150–3155CrossRefPubMedGoogle Scholar
  60. Rotter M, Aprelev A, Adachi K, Ferrone F (2005) Molecular crowding limits the role of fetal hemoglobin in therapy for sickle cell disease. J Mol Biol 347:1015–1023CrossRefPubMedGoogle Scholar
  61. Sasahara K, McPhie P, Minton AP (2003) Effect of dextran on protein stability and conformation attributed to macromolecular crowding. J Mol Biol 326:1227–1237CrossRefPubMedGoogle Scholar
  62. Shastry M, Eftink M (1996) Reversible thermal unfolding of ribonuclease T1 in reverse micelles. Biochemistry 35:4094–4101CrossRefPubMedGoogle Scholar
  63. Shtilerman M, Ding PT, Lansbury PT (2002) Molecular crowding accelerates fibrillization of alpha-synuclein: could an increase in the cytoplasmic protein concentration induce Parkinson’s disease? Biochemistry 41:3855–3860CrossRefPubMedGoogle Scholar
  64. Somero GN, Osmond CB, Bolis CL (1992) Water and life. Springer, BerlinGoogle Scholar
  65. Spencer D, Xu K, Logan T, Zhou H (2005) Effects of pH, salt, and macromolecular crowding on the stability of FK506-binding protein: an integrated experimental and theoretical study. J Mol Biol 351:219–232CrossRefPubMedGoogle Scholar
  66. Steadman BL, Trautman PA, Lawson EQ, Raymond MJ, Mood DA, Thomson JA, Middaugh CR (1989) A differential scanning calorimetric study of the bovine lens crystallins. Biochemistry 28:9653–9658CrossRefPubMedGoogle Scholar
  67. Tellam RL, Sculley MJ, Nichol LW, Wills PR (1983) Influence of polyethylene glycol 6000 on the properties of skeletal-muscle actin. Biochem J 213:651–659PubMedGoogle Scholar
  68. Tokuriki N, Kinjo M, Negi S, Hoshino M, Goto Y, Urabe I, Yomo T (2004) Protein folding by the effects of macromolecular crowding. Protein Sci 13:125–133CrossRefPubMedGoogle Scholar
  69. Uversky V, Cooper M, Bower K, Li J, Fink A (2002) Accelerated alpha synuclein fibrillation in crowded milieu. FEBS Lett 515:99–103CrossRefPubMedGoogle Scholar
  70. van den Berg B, Ellis R, Dobson C (1999) Effects of macromolecular crowding on protein folding and aggregation. EMBO J 18:6927–6933CrossRefPubMedGoogle Scholar
  71. Wilf J, Gladner JA, Minton AP (1985) Acceleration of fibrin gel formation by unrelated proteins. Thromb Res 37:681–688CrossRefPubMedGoogle Scholar
  72. Zhou HX (2004) Protein folding and binding in confined spaces and in crowded solutions. J Mol Recognit 17:368–375CrossRefPubMedGoogle Scholar
  73. Zhou HX, Dill KA (2001) Stabilization of proteins in confined spaces. Biochemistry 40:11289–11293CrossRefPubMedGoogle Scholar
  74. Zhou HX, Rivas G, Minton AP (2008) Annu. Rev. Biophys. 37:375–397Google Scholar
  75. Zimmerman SB, Harrison B (1987) Macromolecular crowding increases binding of DNA polymerase to DNA: an adaptive effect. Proc Natl Acad Sci USA 84:1871–1875CrossRefPubMedGoogle Scholar
  76. Zimmerman SB, Trach SO (1991) Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J Mol Biol 222:599–620CrossRefPubMedGoogle Scholar
  77. Zimmerman SB, Minton AP (1993) Macromolecular crowding: biochemical, biophysical, and physiological consequences. Annu Rev Biophys Biomol Struct 22:27–65CrossRefPubMedGoogle Scholar

Copyright information

© Springer Netherlands 2011

Authors and Affiliations

  1. 1.Section on Physical Biochemistry, Laboratory of Biochemistry and GeneticsNIDDK, National Institutes of HealthBethesdaUSA
  2. 2.Chemical and Physical Biology Program, Centro de Investigaciones BiológicasCSICMadridSpain

Personalised recommendations