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    Infrared Spectroscopy for Protein Secondary Structure Analysis

      The secondary structure of a protein refers to the specific conformation formed by the rotation or folding of the polypeptide backbone atoms along a certain axis, that is, the spatial position arrangement of the polypeptide backbone atoms, which does not involve the amino acid residue side chain. The secondary structure of proteins mainly includes α-helices, β-pleated sheets, β-turns, and random coils, with hydrogen bonds being the main force maintaining these secondary structures.


      The secondary structure of proteins is key to understanding protein function and interactions. There are many methods for detecting the secondary structure of proteins, commonly used ones include Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD), X-ray diffraction, Ultraviolet and Fluorescence Spectroscopy, and Infrared Spectroscopy (IR). These methods all have certain limitations: NMR is suitable for the structure of proteins with relatively small molecular weights. CD can only be used for the detection of clarified protein solutions at low concentrations. X-ray diffraction cannot detect the effect of physiological conditions on the secondary structure of proteins. Fluorescence and UV spectroscopy are limited to the structural analysis of proteins with chromophores (such as Trp, Tyr, etc.). IR, however, is not limited by functional groups, protein state, molecular weight, concentration, environment, and other conditions. In addition, IR uses less sample for determining protein secondary structure, is not affected by light scattering and fluorescence, and can also achieve the study of kinetic properties. At the same time, IR has the advantages of simple operation, high wavelength accuracy, good resolution, fast scanning speed, high sensitivity, etc., and is widely used in various fields of production and scientific research.


      The principle of IR for determining protein secondary structure is based on electromagnetic radiation, with a wavelength range between 4000 and 400 cm-1. The C=O and N-H structures of the peptide bond in proteins will absorb specific wavelengths of infrared light, mainly appearing in the 1700-1600 cm-1 (Amide I band) and 1550-1500 cm-1 (Amide II band) regions of the infrared spectrum. The Amide I band contains rich secondary structure information of proteins, such as α-helix, β-pleated sheet, random coil structure, etc., so the Amide I band is often used to analyze the secondary structure of proteins. By analyzing and fitting the absorption peaks in the Amide I band region, the relative content of various secondary structures in the protein can be obtained.


      MtoZ Biolabs has established the FT-IR analysis platform by using Thermo's Nicolet series of instruments, to measure the infrared spectra of proteins and peptides in samples and perform subsequent baseline correction, Gaussian deconvolution, second-order derivative fitting, and finally determine the secondary structure information of proteins and peptides in samples based on peak area. Contact us for free project consultation.

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