Protein Circular Dichroism Curve Analysis

Protein is a chiral molecule, with the main photoactive groups originating from the peptide bonds in the peptide chain skeleton, aromatic amino acid residues, and disulfide bridges. When plane circularly polarized light passes through these photoactive chromophores, the photoactive centers absorb the left and right circularly polarized light in the plane circularly polarized light differently, creating a difference in absorption. Due to the presence of this absorption difference, the amplitude difference of the polarized light vector is created, and the circularly polarized light becomes elliptically polarized light. This phenomenon is known as the circular dichroism of proteins.

 

Circular dichroism spectrometers can detect this circular dichroism of proteins. If the difference in absorption coefficients Δε=εL- εR is plotted on the y-axis and the wavelength λ of plane polarized light on the x-axis, the resulting graph is a circular dichroism spectrum, also known as a CD spectrum. The CD spectrum of proteins is typically divided into two wavelength ranges: far ultraviolet CD spectrum and near ultraviolet CD spectrum.

 

1. Far Ultraviolet CD Spectrum

The wavelength range of the far ultraviolet region is usually 178～250nm. This region reflects the circular dichroism of the peptide bond. In the regular secondary structure of proteins or polypeptides, the peptide bonds are highly orderly arranged, and the directionality of the arrangement determines the splitting of the peptide bond energy level transitions. Therefore, proteins or polypeptides with different secondary structures produce CD bands with different locations and absorption strengths. The α-helical structure has a positive β band near 192nm, and two negative characteristic shoulder bands at 222 and 208nm; the β-fold CD spectrum has a negative band at 216nm, and a positive band at 185～200nm; the irregular conformation has a negative peak near 198nm, and a small and wide positive peak near 220nm. By using the increase or decrease of the negative wave trough at 208 and 222 nm in the far ultraviolet CD spectrum of proteins, the changes in the α-helix and β-fold content of the protein can be estimated.

 

2. Near-Ultraviolet Circular Dichroism Spectrum

The wavelength range of the near ultraviolet region is usually 250～320nm, and this region is mainly related to the side chain chromophores. For example, the absorption peak of the disulfide bond is at 250～260 nm; the absorption peak of Trp, Tyr and Phe residues is at 230～370nm.

 

MtoZ Biolabs Limited has established a circular dichroism spectrum analysis platform based on the Jasco circular dichroism spectrometer. By combining the use of CDNN software, it can quickly and accurately analyze the secondary and higher-level structure information of proteins/peptides and other chiral molecules. You only need to send samples for a one-stop service: sample processing - machine analysis - data analysis - project report. We look forward to your consultation.