Vibrational Sum-Frequency Scattering for Detailed Studies of Collagen Fibers in Aqueous Environments

Protein fibers play a crucial role in many disease related phenomena and biological systems. A structural analysis of fibrous proteins often requires labeling approaches or disruptive sample preparation while it lacks chemical specificity. Here we demonstrate that the technique of vibrational sum-frequency scattering (SFS) provides a label-free pathway for the chemical and structural analysis of protein fibers in solution. By examining collagen, the most abundant protein in mammals, we demonstrate that the SFS signal of fibers can be detected in the NH, CH stretching and bending, and amide I regions. SFS spectra were found to depend on the scattering angle, which implies the possibility to selectively probe various features of the fibers. The fitting of the data and maximum entropy method analysis revealed a different phase for side-chains and carbonyl contributions, which helps to identify these otherwise overlapping spectral peaks and provides the possibility to perform orientational analysis. Our findings suggest that SFS allows for the greater understanding of protein fibers in solution, which is important when, for example, designing scaffolds in tissue engineering or developing cures for diseases associated with protein fibers.