UV Resonance Raman-Selective Amide Vibrational Enhancement:  Quantitative Methodology for Determining Protein Secondary Structure

• Published in: Biochemistry (Easton) Vol. 37; no. 9; pp. 2854 - 2864
• Main Authors: Chi, Zhenhuan, Chen, X. G, Holtz, Janet S. W, Asher, Sanford A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.03.1998
• Subjects: Amides - chemistry; Circular Dichroism; Models, Chemical; Protein Structure, Secondary; Solutions; Spectrophotometry, Infrared; Spectrum Analysis, Raman - methods; Ultraviolet Rays; Vibration
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi971160z

We have directly determined the amide band resonance Raman spectra of the “average” pure α-helix, β-sheet, and unordered secondary structures by exciting within the amide π→π* transitions at 206.5 nm. The Raman spectra are dominated by the amide bands of the peptide backbone. We have empirically determined the average pure α-helix, β-sheet, and unordered resonance Raman spectra from the amide resonance Raman spectra of 13 proteins with well-known X-ray crystal structures. We demonstrate that we can simultaneously utilize the amide I, II, and III bands and the Cα−H amide bending vibrations of these average secondary structure spectra to directly determine protein secondary structure. The UV Raman method appears to be complementary, and in some cases superior, to the existing methods, such as CD, VCD, and absorption spectroscopy. In addition, the spectra are immune to the light-scattering artifacts that plague CD, VCD, and IR absorption measurements. Thus, it will be possible to examine proteins in micelles and other scattering media.