Circular dichroism (CD)
Circular dichroism (CD) measurements are most commonly used to determine changes in the secondary and tertiary structure of proteins but can be applied to other bio-molecules as well.
The signal obtained from CD measurements (ellipticity) arises from differences in absorption by chiral molecules of left- and right-handed circularly polarized light.
For proteins, Far-UV CD measurements are obtained using circularly polarized light over a wavelength range of about ≤ 200-250 nm. Light absorption in this wavelength range is mainly due to absorption by the peptide bonds. Since each type of secondary structure element (such as α-helix, β-sheet, etc.) has a distinctive spectral CD profile, Far-UV CD is sensitive to changes in the secondary structure. For instance, Far-UV CD measurements can be performed in different solution conditions (pH, excipients, etc.) to determine the effects thereof on the secondary structural content of a given protein. Also, secondary structures can be estimated when compared to library/database information.
Near-UV CD measurements are obtained over a wavelength range of about 250-300 nm, where tyrosine, tryptophan and phenylalanine residues, as well as disulfide bridges absorb light and are CD sensitive. Near-UV CD signals largely depend on the relative orientation as well as the local mobility of these residues. Consequently, Near-UV CD measurements can provide information about changes in the tertiary structure of proteins, e.g., because of formulation conditions, elevated temperature, storage.