Circular dichroism (CD) is a powerful technique for determining the secondary structure of proteins. It measures the difference in absorption of left- and right-handed circularly polarized light by proteins, providing information about their conformation. CD is particularly useful for assessing the secondary structure of proteins obtained via recombinant methods or purified from tissues. It can also be used to study protein interactions and monitor conformational changes due to temperature, mutations, or binding. However, CD does not provide residue-specific information, unlike X-ray crystallography or NMR. CD measurements can be performed on multiple samples containing up to 20 µg of protein in physiological buffers within a few hours. The technique is advantageous for rapid, high-throughput analysis but requires careful preparation of buffers and proteins to ensure accurate results. Buffers must be optically inactive and transparent, and proteins should be at least 95% pure. Protein concentration must be accurately determined, often using methods like amino acid analysis or molar extinction coefficients. CD data analysis involves estimating the secondary structure of proteins using various methods, including linear regression, ridge regression, singular value decomposition, and neural networks. These methods rely on comparing the CD spectrum of a protein to reference spectra of known secondary structural elements. The most accurate results are obtained using constrained least squares fitting, which ensures that the sum of all fractional contributions equals one. The protocol outlines the steps for setting up CD instruments, preparing buffers and proteins, and analyzing CD spectra to estimate secondary structure. It includes detailed instructions for protein concentration determination, sample preparation, and data analysis. The process involves collecting baseline spectra, subtracting baseline corrections, and converting data to mean residue ellipticity or Δε for further analysis. The data is then analyzed using various software tools to estimate the secondary structure of proteins. The protocol also includes troubleshooting tips for common issues encountered during CD measurements.Circular dichroism (CD) is a powerful technique for determining the secondary structure of proteins. It measures the difference in absorption of left- and right-handed circularly polarized light by proteins, providing information about their conformation. CD is particularly useful for assessing the secondary structure of proteins obtained via recombinant methods or purified from tissues. It can also be used to study protein interactions and monitor conformational changes due to temperature, mutations, or binding. However, CD does not provide residue-specific information, unlike X-ray crystallography or NMR. CD measurements can be performed on multiple samples containing up to 20 µg of protein in physiological buffers within a few hours. The technique is advantageous for rapid, high-throughput analysis but requires careful preparation of buffers and proteins to ensure accurate results. Buffers must be optically inactive and transparent, and proteins should be at least 95% pure. Protein concentration must be accurately determined, often using methods like amino acid analysis or molar extinction coefficients. CD data analysis involves estimating the secondary structure of proteins using various methods, including linear regression, ridge regression, singular value decomposition, and neural networks. These methods rely on comparing the CD spectrum of a protein to reference spectra of known secondary structural elements. The most accurate results are obtained using constrained least squares fitting, which ensures that the sum of all fractional contributions equals one. The protocol outlines the steps for setting up CD instruments, preparing buffers and proteins, and analyzing CD spectra to estimate secondary structure. It includes detailed instructions for protein concentration determination, sample preparation, and data analysis. The process involves collecting baseline spectra, subtracting baseline corrections, and converting data to mean residue ellipticity or Δε for further analysis. The data is then analyzed using various software tools to estimate the secondary structure of proteins. The protocol also includes troubleshooting tips for common issues encountered during CD measurements.