Advancements in Mid-Infrared Imaging and Spectroscopic Techniques for the Study of Phase Separating Peptides

Liquid-liquid phase separating (LLPS) proteins have been the center of concentrated research efforts over the past decade due to their relevance in many biological functions and systems, as well as their involvement in various neurodegenerative diseases. While current evidence links condensates formed by LLPS proteins to a number of mechanisms for biological function, the role of these condensates has proven difficult to study directly. Model peptides that undergo phase separation have been designed to help facilitate the study of these systems. It has been proposed that these peptides, when in condensate phase, undergo changes in secondary structure. While research has been done to show that the condensates can cause changes to protein and peptide behavior, work on structural changes is limited, especially in situ. The majority of these studies are computational work or utilize the addition of fluorescent labels or tags. Infrared (IR) spectroscopy and microscopy techniques are uniquely qualified to detect secondary structural changes, among other characteristics, in complex environments. To study these changes, two-dimensional infrared spectroscopy (2DIR) experiments have been done to compare structural differences in bulk solution versus condensate-forming conditions for several model peptide systems. The spectra resulting from these studies, however, do not always show any noticeable differences. While it is plausible in these cases that there is no substantial difference in overall secondary structure between bulk solution and condensate phase for these particular peptide systems, it is also possible that we were unable to collect 2DIR of the condensates specifically. With the use of two-dimensional infrared hyperspectral imaging, secondary structural information can be distinctly gathered on the proteins both within and outside of the LLPS condensates. At this time, however, the technology to perform such an experiment is underdeveloped. Mid-infrared light lacks the ability to measure anything smaller than a few microns due to the classical resolution limit and its long wavelengths. Additionally, the broadband light necessary for 2DIR spectroscopic measurements causes chromatic dispersion, further worsening the actual experimental resolution. The work presented here progresses the instrumental development necessary for super resolution, two-dimensional infrared hyperspectral imaging of liquid-liquid phase separating systems, as well as presents preliminary studies on several model LLPS systems.