Measurement of anisotropy of pore diffusion in protein crystals by PFG NMR and by CLSM

Solvent (water) and solute (fluorescein and rhodamine B) diffusion in crystalline lysozyme protein, which represent a mesoporous material with an anisotropic pore structure, was studied by pulsed field gradient nuclear magnetic resonance (PFG NMR) and confocal laser scanning microscopy (CLSM), respectively. Water and fluorescent dyes reveal anisotropic intracrystalline diffusion behaviour. The averaged intracrystalline self-diffusion coefficient of water was found to be about one order of magnitude smaller than the value for bulk liquid water at the same temperature. However, the intracrystalline self-diffusion coefficients of water are about two orders of magnitude faster than the corresponding values of the fluorescent dyes. For water and fluorescein in tetragonal lysozyme crystals, the anisotropy ratio, which is defined as the ratio of the largest component of the diffusion tensor to its averaged orthogonal value, was about 0.16, whereas for orthorhombic crystals the anisotropy ratios measured by CLSM for fluorescein (0.15 ± 0.03) and rhodamine B (0.25 ± 0.05) were in the same order of magnitude but somewhat larger compared to the value for water (0.11 ± 0.04) measured by PFG NMR.