Circular Dichroism Spectroscopy for Protein Determination

Circular Dichroism (CD) is a spectroscopic measurement based on the optical isomerism of plane-polarized light by optically active substances. When plane-polarized light passes through an optically active substance, its two orthogonal oscillation components are absorbed differently, resulting in elliptically polarized light.

 

CD spectra provide information on the structure and conformational changes of proteins, polypeptides, and other optically active molecules. The secondary structural elements of proteins, such as α-helices, β-sheets, β-turns, and random coils, have specific CD signals. By analyzing CD spectra, the secondary structural components of proteins can be estimated.

 

Analysis Workflow

1. Sample Preparation

Choose a suitable buffer that does not produce a significant background signal within the measurement range of the CD spectrum. The concentration of the protein usually ranges from 0.1 to 1 mg/mL, but the optimal concentration depends on the properties of the protein and the sensitivity of the CD spectrometer.

 

2. Background Measurement

Measure the CD signal of the selected buffer without the protein to obtain the background spectrum.

 

3. Sample Measurement

Measure the CD signal of the protein sample in the range of about 190-250 nm.

 

4. Data Processing

Subtract the background spectrum from the CD signal of the protein sample. Convert the resulting differential spectrum to mean residue ellipticity (MRE), a unit related to protein concentration and path length.

 

5. Data Interpretation

Estimate the content of the protein's secondary structure based on the characteristic wavelengths and shapes of the spectrum. More accurate calculations of the proportions of α-helices, β-sheets, random coils, and other secondary structures can be made using specialized software (such as SELCON3, CONTIN, or CDPro).

 

The advantages of CD determination are that it is fast, non-destructive to the sample, and can be used for real-time monitoring of protein conformational changes. However, it should be noted that CD spectra can only provide approximate information about the secondary structure of proteins, not detailed three-dimensional structural information.