22] Stopped-flow circular dichroism

Abstract

This chapter discusses the use of Stopped-Flow Circular Dichroism (SFCD) in studying protein conformational changes, and in particular, its application in the investigation of the mechanism of protein folding and assembly. SFCD is the adaptation of stopped flow mixing techniques to the measurement of circular dichroism The CD in the farultraviolet peptide-absorbing regions (200-240 nm) is, for the most part, because of the secondary structure of the polypeptide chain, so that the formation of such structures in folding reactions can be isolated, independent of other levels of structure. Thus, while time-dependent changes in optical activity of various side chains or prosthetic groups (in the near-ultraviolet and visible regions) may reveal specific aspects of the folding process, farultraviolet SFCD is potentially a more powerful tool in that it will almost always yield a directly interpretable result, namely the time-course of secondary structure formation. A multiprobe analysis of a folding reaction, which includes SFCD together with various probes of tertiary structure, should therefore be of decisive help in unraveling the details of protein folding.