Circular Dichroism in Analysis of Biomolecules
Authors: Rodger Alison , Pančoška Petr 
Published: September 2024

AbstractCircular dichroism(CD) spectroscopy belongs to the family of chiroptical methods. These methods utilize the interaction ofcircularly polarized light(CPL) with chiral molecules and molecular systems to obtain information about their structure and electronic or vibrational states. A CD spectrum Δϵ(ν) is the difference of two absorbance spectra: one measured withleft circularly polarized light(LCPL) and the other one recorded withright circularly polarized light(RCPL) Δϵ(ν) = ϵL(ν)–ϵR(ν). CD spectra can be measured aselectronic circular dichroism(ECD) in spectral regions of electronic transitions (ultraviolet(UV) andvisible(VIS) light) and asvibrational circular dichroism(VCD) in theinfrared(IR) spectral regions. An ECD spectrum for chiral fluorophores can be also recorded as the difference excitation spectrum for fluorescence spectra excited by LCPL and RCPL, respectively (fluorescence‐detected circular dichroism(FDCD)).Raman optical activity(ROA) and circularly polarized luminescence spectroscopies complete the family of currently developed chiroptical methods. The differences Δϵ(ν) measured as CD are typically ∼10−5of sample absorbance. Special modifications of dispersive (for ECD and VCD) orFourier transform infrared(FTIR) (for VCD) spectrometers are needed to measure CD with a reasonablesignal‐to‐noise ratio(S/N). Polarization modulation of the incident light by a photoelastic modulator and synchronous electronic processing of the resulting photoelectric signal in the spectrometers are typically used for this purpose.Molecular structure is encoded in the CD spectra because the chiral field of the CPL wave that induces a spectral transition in the chiral molecule can be observably altered both by the transition electron density redistribution (as in conventional absorption spectroscopy) and by the transition magnetic field accompanying the molecular charge redistribution. The structure and conformation of the studied molecule define the relative orientation of the characteristic directions of these two effects. This relative orientation affects the probability of absorption of photons of RCPL and LCPL and, in turn, determines the CD sign, the primary information that is unique for CD spectroscopy. In the absolute value, CD intensity (“secondary” unique information) is related to the angle of electric and magnetic transition moments and can, therefore, be converted into the molecular conformation once a suitable theoretical model is available. For complicated molecules (biomolecules, proteins, nucleic acids) where the corresponding theoretical calculations are too complex, empirical interpretive methods based on reference sets of spectra measured for molecules with known structures (from X‐ray ornuclear magnetic resonance(NMR) or cryo‐electron microscopy experiments) are used with success. For example, using these empirical methods, the fractions of regular secondary structures (secondary structure fraction(SSF)) in globular proteins can be determined with a relative error of 3–7% and the number of secondary structure segments in proteins with a typical error of one to three segments per protein fold.CD spectroscopy is highly sensitive to polarization artifacts that can be related to optical imperfections of the optical parts of the spectrometer. To achieve an acceptable S/N, the sample total absorbance A should be also controlled (typically A < 1). This, in effect, restricts the selection of usable solvents and largely determines the concentration and/or path length ranges of studied samples.