Circular and Linear Dichroism Spectroscopy for the Study of Protein–Ligand Interactions

Abstract

Circular dichroism (CD) is the difference in absorption of left and right circularly polarized light, usually by a solution containing the molecules of interest. A non-zero signal for solutions is only measured for chiral molecules such as proteins whose mirror image is not superposable on the original molecule. A CD spectrum provides information about the bonds and structures responsible for the chirality. When a small molecule (or ligand) binds to a protein, it acquires an induced CD (ICD) spectrum through chiral perturbation to its structure or electron rearrangements (transitions). The wavelengths of this ICD are determined by the ligand’s own absorption spectrum, and the intensity of the ICD spectrum is determined by the strength and geometry of its interaction with the protein. Thus, ICD can be used to probe the binding of ligands to proteins. This chapter contains an outline of how to perform protein CD and ICD experiments, together with some of the issues relating to experimental design and implementation. Addition of a quarter wave plate to a CD spectropolarimeter converts it to a linear dichroism (LD) spectrometer. When protein samples are aligned either in flow (as for fibers or membrane proteins in liposomes) or on surfaces the orientations of ligands with respect to the protein backbone or other subunits can be determined.