Protein crystals are widely used for structural determination using X-ray crystallography and solid state NMR. X-ray free electron lasers have enabled direct dynamical structural movies of photo or chemically initiated biochemical reactions. However the perturbation of protein dynamics by the crystal contact forces has been persistently under debate. If crystal contact forces are sufficiently strong, the time resolved X-ray diffraction measurements will not accurately reflect the in vivo biological dynamics. Here we probe the effect of crystal contact forces on protein structural dynamics by directly measuring the collective intramolecular vibrations as a function of crystal symmetry for chicken egg white lysozyme (CEWL). These vibrations extend throughout the protein, involving coherent motion of entire domains. While the collective vibrational density of states is a broad continuous spectrum peaked near 100 cm−1, (3 THz), polarization dependent terahertz absorption isolates vibrations with displacements primarily along the polarization direction [1]. Triclinic, monoclinic, tetragonal and orthorhombic CEWL crystals are grown according to previous published protocols. X-ray measurements verify their crystal structure and quality before and after terahertz measurements. The anisotropic terahertz absorbance is measured using our new technique: ideal polarization varying anisotropic THz microscopy (IPV-ATM). This near field THz microscopy method allows rapid determination of the anisotropic THz absorption. By starting from measurements of the lowest symmetry, triclinic crystals, which have a single protein per unit cell and absolute alignment of the protein molecule array, we can predict the spectra for higher symmetry groups, neglecting crystal contact perturbations. We compare our predicted spectra to the measurements of the monoclinic, orthorhombic and tetragonal crystals, attaining a measure of the impact of the intermolecular crystal forces on the intramolecular vibrations. [1]G. Acbas, K. A. Niessen, E. H. Snell, and A. G. Markelz, Nat. Comm.(2014) https://doi.org/10.1038/ncomms4076.
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