Near-Infrared and Mid-Infrared Fourier Transform Vibrational Circular Dichroism of Proteins in Aqueous Solution

Abstract

Vibrational circular dichroism (VCD) of a series of proteins in H₂O solution with differing secondary structure are reported for the first time in the near-infrared (NIR) region as well as the NH-stretching region. The Fourier transform (FT) near-infrared (NIR) measurements were carried out between 6000 to 4000 cm⁻¹. FT-VCD measurements were simultaneously carried out for the mid-infrared (mid-IR) region from 2000 to 800 cm⁻¹ for direct comparison to VCD in the NIR region. The NIR VCD spectra of proteins show distinct spectral features for different protein structural motifs, indicating a valuable new method to study protein conformations. The principal VCD transitions in the NIR region are two combination bands, the amide A-II and B-II bands, of the amide A and B fundamentals with the amide II fundamental, and the second overtone of the amide II, referred to as the amide 3 X II band. VCD in the amide A and B band region consisting primarily of NH stretching motions were successfully obtained in H₂O for the first time for an insulin fibril sample. Similar to the enhanced VCD signal observed in amide I and II regions, the amide A and B VCD of insulin fibril shows strong intensity enhancements, providing an additional valuable probe of protein fibril growth and development in solution. The relative sensitivities of the mid-IR, N–H stretching, and NIR regions are discussed.