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

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.