What Is the Principle of Infrared Spectroscopy in Protein Secondary Structure Analysis?
Infrared (IR) spectroscopy is an analytical technique based on electromagnetic radiation with a wavelength range of 4000–400 cm⁻¹. In proteins, the C=O and N-H groups in peptide bonds exhibit characteristic infrared absorption, primarily in the 1700–1600 cm⁻¹ region (Amide I band) and the 1550–1500 cm⁻¹ region (Amide II band). The specific infrared absorption frequencies of these vibrational modes are influenced by the surrounding molecular environment. Different protein secondary structures, such as α-helices, β-sheets, and random coils, lead to slight shifts in the absorption frequencies of the C=O and N-H vibrations.
By analyzing and fitting the absorption peaks in the Amide I band, the relative proportions of various secondary structures within a protein can be quantified. To improve spectral resolution and signal-to-noise ratio, deuterium oxide (D₂O) is often used as a solvent instead of regular water. In D₂O, the strong infrared absorption of water is shifted, preventing interference with protein absorption signals and enabling clearer detection of protein infrared spectra.
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