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.

Characterization of heme binding to recombinant α1-microglobulin

“…The induced CE observed for heme/A1M samples were compared with those measured for heme/HPX and heme/HA samples of the same heme-to-protein molar ratio (Figure 6C ). Although the three proteins are quite different, the intensity of the induced CD is generally determined by the strength of heme interactions with each protein and geometry of the binding site (Daviter et al, 2013 ). Therefore, higher intensities of the induced CD spectra in case of heme/HPX and heme/HA, in comparison with that of heme/A1M, suggest that heme binding sites in hemopexin and albumin are tighter and of higher affinity than those of A1M.…”

PDZ Sample Quality Assessment by Biochemical and Biophysical Characterizations

“…However, it is more sampleand time-consuming than SEC SLS and will require a specialist help. An analytical ultracentrifuge allows monitoring the sedimentation process of a sample while moving in the centrifugal field [23]. It will separate PDZ domains according to their sedimentation features which are linked to their size and molecular mass.…”

Spectroscopy of model-membrane liposome-protein systems: complementarity of linear dichroism, circular dichroism, fluorescence and SERS

“…With targeted labelling, fluorescence can indicate the depth of a protein in the membrane, as well as giving an indication of protein and lipid dynamics [6]. Circular dichroism (CD) spectroscopy quickly indicates whether a peptide or protein has changed conformation [7] and linear dichroism (LD) [8] can be used to determine the orientation of protein secondary structures with respect to the membrane normal. These spectroscopies are usually implemented with UV-visible light.…”

“…Figure8. FDLD of M13 bacteriophage aligned in Couette flow (0.5 mm path length) (bacteriophages depicted on the left and FDLD spectra on the right)[69] showing the relatively larger magnitude signals for the lowest electronic state of tryptophans as found in standard fluorescence measurements.Data were collected with a Jasco J-815 spectropolarimeter adapted for LD with Semrock 300 nm long-pass edge filter inserted before the detector to block transmitted incident light.…”