Protein Unfolding and Denaturants

Abstract

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 ΔG0H0TΔS0.

  • 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 . 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 , and . (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.

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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]