Protein Unfolding and Denaturants

Lars Konermann, The University of Western Ontario, London, Ontario, Canada

Published online: May 2012

DOI: 10.1002/9780470015902.a0003004.pub2

The biologically active (native) state of most proteins is characterised by a tightly folded and highly ordered conformation. Denaturants are chemical or physical agents that can induce unfolding of the polypeptide chain. Examples include urea, heat, extremes of pH, as well as some detergents. Unfolded proteins adopt a largely disordered structure. Denaturants can interact directly with the protein, or they can alter the properties of the surrounding aqueous environment. Despite the routine use of denaturants in the biochemical laboratory, the mechanisms whereby these agents destabilise the native state remain poorly understood. Folded protein structures are only marginally stable. As a result, relatively subtle alterations in the physical and chemical properties of the solvent can cause major changes in position of the unfolding equilibrium. This review briefly discusses the most commonly used denaturants, their likely mechanisms of action, as well as some thermodynamic aspects.

Key Concepts:

  • The biologically active (native) state N of proteins represents a highly ordered and tightly folded structure.
  • N is in equilibrium with an extensively disordered (unfolded) state U.
  • Denaturing agents (e.g. urea, extremes of pH and temperature) shift the unfolding equilibrium from N to U.
  • The exact mechanism of action remains unclear for most denaturants.
  • The position of the unfolding equilibrium is governed by an interplay of enthalpic and entropic contributions that affect the free energy of unfolding according to ΔG0=ΔH0–TΔS0.
  • The sign and magnitude of ΔG0 represents the thermodynamic stability of N; N is stable when ΔG0>0.
  • Unfolding can be triggered by increasing the temperature T, because U has a higher entropy than N (ΔS0>0).
  • Protein stability measurements are commonly carried out by employing urea or guanidinium chloride‐induced unfolding in conjunction with optical detection.
  • Some proteins adopt semiunfolded structures (intermediates) under mildly denaturing conditions.
  • Protein (un)folding can be studied under equilibrium conditions and in kinetic experiments.

Keywords: protein folding; protein denaturation; protein conformation; folding intermediate; thermodynamics

Figure 1. Schematic representation of a reversible two‐state protein unfolding equilibrium. The native state (N) corresponds to a highly ordered and tightly folded conformation; the unfolded state (U) represents a largely disordered structure. The equilibrium constant Keq=[U]/[N] depends on the denaturant concentration in the solvent. In the absence of denaturants, Keq≪1.
Figure 2. Guanidinium chloride and urea represent the two most commonly used protein denaturants.
Figure 3. (a) Plot of the free energy of unfolding (ΔG0) as a function of denaturant concentration [D], based on eqn (6). The following parameters were used: ΔG0water=20 kJ mol−1; m=4 kJ mol−1 M−1; T=295 K. (b) Fraction of unfolded protein f=[U]/([U]+[N]) as a function of [D], calculated from the ΔG0 values depicted in (a). For [D]50=5 M, ΔG0 becomes zero and therefore [U]=[N]. Additional information is given in the text.
Figure 4. (a) Plot of the free energy of unfolding (ΔG0) as a function of temperature, based on eqns (3), (8) and (9). (b) Fraction of unfolded protein, calculated from the free energy profile in (a). Parameters used: ΔCP=3.2 kJ mol−1 K−1, ΔH0R=170 kJ mol−1 and ΔS0R=0.46 kJ mol−1 K−1. Here, the melting temperature of Tm=370 K is identical to the reference temperature TR. The temperature of cold unfolding under these conditions is Tc=273.5 K.
close
 References
    Akiyama S, Takashi S, Ishimori K and Morishima I (2000) Stepwise formation of a‐helices during cytochrome c folding. Nature Structural Biology 6: 514–520.
    Anfinsen CB (1973) Principles that govern the folding of protein chains. Science 181: 223–230.
    Ball P (2011) More than a bystander. Nature 478: 467–468.
    Bartlett AI and Radford SE (2009) An expanding arsenal of experimental methods yields an explosion of insights into protein folding mechanisms. Nature Structural & Molecular Biology 16: 582–588.
    Booth PJ and Clarke J (2010) Membrane protein folding makes the transition. Proceedings of the National Academy of Sciences of the USA 107: 3947–3948.
    Brockwell DJ and Radford SE (2007) Intermediates: ubiquitous species on folding energy landscapes? Current Opinion in Structural Biology 17: 30–37.
    Chattopadhyay K, Elson EL and Frieden C (2005) The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods. Proceedings of the National Academy of Sciences of the USA 102: 2385–2389.
    Dempsey AC, Walsh MP and Shaw GS (2003) Unmasking the annexin I interaction from the structure of apo‐S100A11. Structure 11: 887–897.
    Dobson CM (2003) Protein folding and misfolding. Nature 426: 884–890.
    England JL, Pande VS and Haran G (2008) Chemical denaturants inhibit the onset of dewetting. Journal of the American Chemical Society 130: 11854–11855.
    book Gruebele M (2005) "Fast relaxation methods". In: Buchner J and Kiefhaber T (eds) Protein Folding Handbook, pp. 454–490. New York: Wiley.
    Hughson FM, Wright PE and Baldwin RL (1990) Structural characterisation of a partly folded apomyoglobin intermediate. Science 249: 1544–1548.
    Konermann L, Pan J and Liu Y (2011) Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chemical Society Reviews 40: 1224–1234.
    Lim WK, Rosgen J and Englander SW (2009) Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group. Proceedings of the National Academy of Sciences of the USA 106: 2595–2600.
    Michaux C, Pomroy NC and Prive GG (2008) Refolding SDS‐denatured proteins by the addition of amphipathic cosolvents. Journal of Molecular Biology 375: 1477–1488.
    Myers JK, Pace CN and Schotz JM (1995) Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Science 4: 2138–2148.
    Privalov PL and Khechinashvili NN (1974) A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. Journal of Molecular Biology 86: 665–684.
    Raschke TM and Marqusee S (1997) The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules under native conditions. Nature Structural Biology 4: 298–304.
    Rhoades E, Cohen M, Schuler B and Haran G (2004) Two‐state folding observed in individual molecules. Journal of the American Chemical Society 126: 14686–14687.
    Sawle L and Ghosh K (2011) How do thermophilic proteins and proteomes withstand high temperature? Biophysics Journal 101: 217–227.
    Service RF (2008) Problem solved* (*sort of). Science 321: 784–786.
    Southall NT, Dill KA and Haymett ADJ (2002) A view of the hydrophobic effect. Journal of Physical Chemistry B 106: 521–533.
    Tani F, Shirai N, Onishi T et al. (1997) Temperature control for kinetic refolding of heat‐denatured ovalbumin. Protein Science 6: 1491–1502.
    Thirumalai D and Reddy G (2011) Are native proteins metastable? Nature Chemistry 3: 910–911.
    Zhou Z and Bai Y (2007) Analysis of protein folding cooperativity. Nature 445: E16–E17.
 Further Reading
    Bissantz C, Kuhn B and Stahl M (2010) A medicinal chemist's guide to molecular interactions. Journal of Medicinal Chemistry 53: 5061–5084.
    Chandler D (2007) Oil in troubled waters. Nature 445: 831–832.
    book Creighton TE (1993) Proteins. New York: Freeman.
    book Dill KA and Bromberg S (2003) Molecular Driving Forces. New York: Garland Science.
    book Fersht A (1999) Structure and Mechanism in Protein Science. New York: Freeman.
    Guinn EJ, Pegram LM, Capp MW, Pollock MN and Record MT (2011) Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer. Proceedings of the National Academy of Sciences of the USA 108: 16932–16937.
    book Kaltashov IA and Eyles SJ (2005) Mass Spectrometry in Biophysics. Hoboken, NJ: John Wiley and Sons.
    Konermann L (2006) Exploring the relationship between funneled energy landscapes and two‐state protein folding. Proteins 65: 153–163.
    book Murphy KP (ed.) (2001) Protein Structure, Stability, and Folding. Totowa NJ: Humana Press.
    book Pain RH (ed.) (2000) Mechanisms of Protein Folding. Oxford: University Press.
    Renthal R (2006) An unfolding story of helical transmembrane proteins. Biochemistry 45: 14559–14566.

Related Sub-Topics:

Protein Folding, Evolution and Degradation | Proteins: Structure, Function, Metabolism
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Protein Unfolding and Denaturants
 
How to Cite close
Konermann, Lars(May 2012) Protein Unfolding and Denaturants. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003004.pub2]