Site-specific dichroism analysis utilizing transmission FTIR

Infrared spectroscopy has long been used to examine the average secondary structure and orientation of membrane proteins. With the recent utilization of site-specific isotope labeling (e.g., peptidic 1-¹³C = ¹⁸O) it is now possible to examine localized properties, rather than global averages. The technique of site-specific infrared dichroism (SSID) capitalized on this fact, and derives site-specific orientational restraints for the labeled amino acids. These restraints can then be used to solve the backbone structure of simple α-helical bundles, emphasizing the capabilities of this approach. So far SSID has been carried out in attenuated total internal reflection optical mode, with all of the respective caveats of attenuated total internal reflection. In this report we extend SSID through the use of transmission infrared spectroscopy tilt series. We develop the corresponding theory and demonstrate that accurate site-specific orientational restraints can be derived from a simple transmission experiment.