We are using THz Time-Domain Spectroscopy (THz-TDS) and Microscopy to measure correlated vibrational modes in proteins. Our technique, which we call Crystal Anisotropy THz Microscopy (CATM), has proven to be successful in measuring vibrational modes in molecular crystals. This technique for the first time enables benchtop measurement and identification of large scale protein vibrational modes, which are widely accepted to be related to protein function and binding. These measurements have several advantages over other techniques, such as neutron and x-ray scattering. First, by using an optical technique we select only those modes with a high dipole coupling. Secondly these measurements are made at room temperature, with full hydration, on a table top with small sample requirements. Our THz microscope is based on the design of Planken et al. Due to the small size of the protein crystals (~300 microns), the measurements are done in the near field to overcome the THz diffraction limit.
The figure shown is an example of our measurements of a single orientation of a chicken egg-white lysozyme (CEWL) crystal. The horizontal axis shows the crystal orientation in degrees relative to a single crystal orientation and the terahertz frequency. The vertical scale represents the relative change in absorption for each orientation relative to a reference orientation taken to be zero degrees. Our CEWL measurements compare very well to the calculated response and are the first measurements in which mode identification is possible. We have also measured CEWL bound to the triacetylglucosamine inhibitor (CEWL-3NAG) and see a change in the vibrational modes with an increase in activity at lower frequencies when compared to the CEWL measurements. This result is in agreement with a higher density of states at lower frequency with binding.
Our measurements and CATM technique are the first which have shown that protein mode identification is possible. The changes in these modes with binding are consistent with what we would expect and open the door for protein binding or protein mutation to identify protein functional mechanisms, important for protein engineering for biomedical and technological applications.
Resources For THz Optics: