Protein Denaturation and the Denatured State

Abstract

Protein denaturation experiments are routinely used to determine protein stability and to elucidate structural and dynamic effects of mutations, cofactors and ligands. Denatured states of proteins have gained wide interest in recent years owing to their fundamental importance in a wide variety of phenomena such as deciphering the protein folding problem and the molecular understanding of many diseases. Because the integrity of the folded protein structure is strongly affected by environmental factors denaturation is often studied by monitoring the effects of increasing temperature, changing pH or addition of chaotropic agents such as urea and guanidinium chloride. The structural and dynamical effects of denaturation are most efficiently characterised by using a combination of spectroscopic methods most notably circular dichroism, fluorescence and nuclear magnetic resonance. Amide hydrogen–deuterium exchange experiments can efficiently characterise partly unfolded protein structures.

Key Concepts:

  • There are many spectroscopic methods to study protein denaturation and denatured states.

  • Transitions between folded and denatured conformations of proteins are often highly cooperative.

  • Denatured states of proteins are ensembles of rapidly converting conformations on the nanosecond timescale.

  • Denatured states of proteins possess residual structures which often reside within hydrophobic cores.

  • Denatured states of proteins can be informative of the protein folding pathway.

Keywords: intermediate; partly unfolded conformations; molten globule; residual structure; misfolding; protein compactness; fluorescent probes; site‐directed labelling

Figure 1.

Schematic of protein denaturation showing protein cartoons of conformational states and varying degrees of cooperativity in the transitions between them. Protein compactness is plotted versus denaturation stress (e.g. increasing concentration of denaturant (GuHCl, urea, guanidinium isothiocyanate) or increasing temperature or decreasing pH). The denaturation curve is shown in red. Blue cartoons represent conformational states of protein molecules in native (N), intermediate (I) (including molten globules), Uc and Ue. The transitions between these conformational states are numbered 1 to 3, in decreasing order of cooperativity. Clearly, the most cooperative transition is 1 between N and I, followed by the unfolding of I to Uc. Transition 3 describes the noncooperative unfolding of the compact unfolded state.

Figure 2.

Energy landscape of protein denaturation shown as a ‘funnel’. The width of the funnel represents the conformational heterogeneity of the protein and the energy represents the height of the funnel under folding‐permissive conditions (e.g. physiological conditions). The conformational states of Figure are placed at various positions inside the funnel. The most ordered conformation, having the lowest energy (most stable) under physiological conditions, is the well‐defined structure of the native state (N). The most heterogeneous states are represented by the expanded unfolded states (Ue). Reproduced with permission from Dill and Chan . © Nature Publishing Group.

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Reference

Dill KA and Chan HS (1997) From Levinthal to pathways to funnels. Nature Structural Biology 4: 10–19.

Further Reading

Bowler BE (2012) Residual structure in unfolded proteins. Current Opinion in Structural Biology 22(1): 4–13.

Fersht AR (1999) Structure and Mechanism in Protein Science, pp 508–614. New York: WH Freeman.

Gross M (2000) Proteins that convert from alpha helix to beta sheet: implications for folding and disease. Current Protein and Peptide Science 1: 339–347.

Hammarström P and Carlsson U (2000) Is the unfolded state the Rosetta Stone of the protein folding problem? Biochemical and Biophysical Research Communications 276: 393–398.

Kjellsson A, Sethson I and Jonsson BH (2003) Hydrogen exchange in a large 29 kD protein and characterization of molten globule aggregation by NMR. Biochemistry 42: 363–374.

Matouschek A (2003) Protein unfolding – An important process in vivo? Current Opinion in Structural Biology 13: 98–109.

Otzen DE and Oliveberg M (1999) Salt‐induced detour through compact regions of the protein folding landscape. Proceedings of the National Academy of Sciences of the USA 96: 11746–11751.

Shortle D (1996) The denatured state (the other half of the folding equation) and its role in protein stability. FASEB Journal 10: 27–34.

Shortle D and Ackerman MS (2001) Persistence of native‐like topology in a denatured protein in 8 M urea. Science 293: 487–489.

Smith LJ, Fiebig KM, Schwalbe H and Dobson CM (1996) The concept of a random coil. Residual structure in peptides and denatured proteins. Folding and Design 1: R95–106.

Uversky VN (2002) Natively unfolded proteins: a point where biology waits for physics. Protein Science 4: 739–756.

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How to Cite close
Hammarström, Per, and Jonsson, Bengt‐Harald(Sep 2013) Protein Denaturation and the Denatured State. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003003.pub2]