Estimation of Protein Secondary Structure from Circular Dichroism Spectra: Inclusion of Denatured Proteins with Native Proteins in the Analysis

Optimized stability retention of a monoclonal antibody in the PLGA nanoparticles

“…The far-UV spectrum of CD generally reflects secondary structures of protein (Sreerama et al, 1999;Johnson, 1999). Various empirical methods and analyses have been developed for quantitative estimation of the secondary structure content of protein (Bohm et al, 1992;Sreerama et al, 2000;Fink, 1995). Effects of sugars, such as mannitol, sucrose and trehalose, on protein stability in emulsion are controversial (Sah, 1999a,b;Kang and Singh, 2003).…”

“…CD spectra were recorded in an average of 10 scans between 190 and 260 nm, with a 1.0 nm bandwidth, a scanning rate of 10 nm/min, a wavelength step of 0.2 nm and a time constant of 2 s. CD band intensities were expressed as molar ellipticities, [Â] M, in degrees cm 2 dmol −1 . The extents of secondary structures of 3D8 scFv were determined from the ellipticities using K2d software (Sreerama et al, 2000(Sreerama et al, , 1999Johnson et al, 1999;Bohm et al, 1992). Fluorescence emission spectra were obtained using a spectrofluorometer Japan) to analyze tertiary structures of recovered 3D8 scFv.…”

CTCF terminal segments are unstructured

“…The accuracy of this calculation should be regarded carefully because many caveats complicate the estimation of unordered content. 25,26 The rough estimate, however, is clear and suggests that the bulk of both terminal fragments are unordered. Unfolded polypeptides are extended and likely account for the large hydrodynamic radii observed.…”

Circular Dichroism and Its Application to the Study of Biomolecules

“…In addition to the assumption of additivity (see Eq. (5)) these methods depend on four other major assumptions (Sreerama et al, 2000): (1) that the effect of tertiary structure on CD is negligible, (2) that the ensemble-averaged solution structure is equivalent to the X-ray structure, (3) that the contributions from non-peptide chromophores do not influence the analysis, and (4) that the geometric variability of secondary structures need not be explicitly considered. Although assumptions (1) and (2) are probably valid, assumptions (3) and (4) are probably invalid in many cases (Sreerama et al, 2000).…”