FAST PROTEIN STRUCTURAL ASSESSMENT USING CIRCULAR DICHROISM (CD) SPECTROSCOPY: AN UPDATED REVIEW

Circular dichroism (CD) spectroscopy has become a rapid and versatile analytical technique for probing protein structure, playing a pivotal role in modern proteomics and structural genomics. This review integrates studies published between 1994-2025 to examine the principles, instrumentation, and diverse applications of CD spectroscopy. CD measures the differential absorption of left-and right-circularly polarized light by chiral molecules, enabling semi-quantitative estimation of protein secondary structures such as α-helices, β-sheets, and random coils using far-UV spectra, and providing insights into tertiary environments via near-UV signals. Its applications include monitoring protein folding and unfolding, characterizing intrinsically disordered proteins (IDPs), and assessing structural integrity in recombinant proteins and biopharmaceuticals. The combination of experimental CD data with computational tools, including vibrational CD (VCD) simulations, the BeStSel algorithm, and machine learning approaches, has improved accuracy, particularly for β-rich, flexible, or disordered protein systems. Despite limitations, such as the need for high-purity samples and approximate secondary structure estimation, CD’s minimal sample requirement, rapid data acquisition, and solution-based analysis make it an essential tool for initial structural evaluation. Looking forward, advances in computational integration, standardized reference databases, and expanded applications to dynamic protein complexes and challenging biomolecules promise to further enhance CD spectroscopy as a cornerstone technique in structural biology and biotechnology.