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Analysis of the kinetics of folding of proteins and peptides using circular dichroism

Nature Protocols volume 1, pages 2891–2899 (2006)Cite this article

Abstract

Circular dichroism (CD) is a useful spectroscopic technique for studying the secondary structure, folding and binding properties of proteins. This protocol covers how to use the intrinsic circular dichroic properties of proteins to follow their folding and unfolding as a function of time. Included are methods of obtaining data and for analyzing the folding and unfolding data to determine the rate constants and the order of the folding and unfolding reactions. The protocol focuses on the use of CD to follow folding when it is relatively slow, on the order of minutes to days. The methods for analyzing the data, however, can also be applied to data collected with a CD machine equipped with stopped-flow accessories in the range of milliseconds to seconds and folding analyzed by other spectroscopic methods including changes in absorption or fluorescence spectra as a function of time.

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Figure 1: Circular dichroism (CD) spectra of proteins and peptides with representative secondary structures.
Figure 2: Circular dichroism of the wild-type (WT) and G14A mutant collagen peptides as a function of temperature and peptide concentration.
Figure 3: Chevron plots of the rate constants of folding and unfolding of two isolated domains of a ribosomal protein, L9, as a function of the concentration of a denaturant.
Figure 4: Unfolding of the wild-type (WT) and G14 mutant collagen peptides as a function of time.
Figure 5: Folding of the wild-type (WT) and G14A mutant collagen model peptides as a function of time.
Figure 6: Linear analyses of the kinetics of folding of the wild-type (WT) collagen peptide at 2 °C.

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Acknowledgements

The research was supported by National Institutes of Health grant GM-36326 to N.J.G. and Sarah E. Hitchcock-DeGregori, and by the Circular Dichroism Facility at Robert Wood Johnson Medical School (UMDNJ). In addition, I thank my collaborators and friends, Barbara Brodsky, Gaetano T. Montelione and especially Sarah E. Hitchcock-DeGregori for their encouragement and support of my studies of protein folding and interactions using CD and NMR.

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  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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Correspondence to Norma J Greenfield.

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Supplementary Equations

Equations for determining rate constants from CD data (PDF 11 kb)

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Greenfield, N. Analysis of the kinetics of folding of proteins and peptides using circular dichroism. Nat Protoc 1, 2891–2899 (2006). https://doi.org/10.1038/nprot.2006.244

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