Determination of Protein Secondary Structure

Abstract

Circular dichroism (CD) is one of the most sensitive physical techniques for determining structures and monitoring structural changes of biomolecules. It can directly interpret the changes of protein secondary structure, even though the method is empirical. The far-ultraviolet (far-UV) CD specra (below 250 nm) of proteins are extremely sensitive toward protein structure, and the near-UV spectra reflect the contributions of aromatic side chains, disulfide bonds, and induced CD bands of prosthetic groups. Together these measurements provide information about the overall structure of a protein molecule as well as its local conformation around the aromatic and prosthetic groups and disulfide linkages. The ease of CD measurements is attractive, but CD, unlike two other powerful techniques—x-ray diffraction of protein crystals and NMR for protein solutions—cannot determine the three-dimensional structure of a protein. In this chapter we will discuss several methods of CD analysis of proteins, which can provide estimates of α helix, β sheet, β turn, and unordered form. These empirical methods utilize a set of reference proteins of known structure from x-ray diffraction studies. Thus, proteins are presumed to have the same structure in the crystalline state and in aqueous solution.