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Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

Nature Protocols volume 1, pages 2527–2535 (2006)Cite this article

Abstract

Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability. If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy and entropy of unfolding, the midpoint of the unfolding transition and the free energy of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength requires ∼8 h.

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Figure 1: CD spectra of polypeptides and proteins with some representative secondary structures.
Figure 2: Spectra of unbound tropomyosin and troponin model proteins and protein-protein complexes.
Figure 3: Typical curves of ellipticity as a function of temperature used to determine the thermodynamics of folding.
Figure 4: Example of the analysis of set of spectra using the CCA.

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Acknowledgements

The work was supported by a National Institutes of Health grant, GM-36326 to N.J.G. and Dr. Sarah E. Hitchcock-DeGregori, and by the Circular Dichroism Facility at Robert Wood Johnson Medical School (UMDNJ). I thank the late Dr. Gerald D. Fasman for supplying copies of programs to deconvolute data sets using the CCA and Dr. Takashi Konno for supplying a SVD program.

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Authors and Affiliations

  1. Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, 08854-5635, New Jersey, USA

    Norma J Greenfield

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  1. Norma J Greenfield
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Correspondence to Norma J Greenfield.

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Supplementary information

Supplementary Equations

Equations for determining the midpoint of a unfolding or folding transition, TM, and enthalpy of folding, ΔH, from changes in ellipticity as a function of temperature. (PDF 45 kb)

Supplementary Tutorial

Deconvolution of a set of curves with the convex constraint algorithm. (PDF 26 kb)

Supplementary Program

DOS version of the CCA for deconvoluting sets of CD spectra. (ZIP 101 kb)

Supplementary Program (use if having trouble unzipping Supplementary Program above)

DOS version of the CCA for deconvoluting sets of CD spectra. (EXE 148 kb)

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Greenfield, N. Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc 1, 2527–2535 (2006). https://doi.org/10.1038/nprot.2006.204

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