Comparing protein-ligand interactions in solution and single crystals by Raman spectroscopy

By using a Raman microscope, we show that it is possible to probe the confo rmational states in protein crystals and crystal fragments under growth con ditions (in hanging drops). The flavin cofactor in the enzyme para-hydroxyb enzoate hydroxylase can assume two conformations: buried in the protein mat rix ("in") or essentially solvent-exposed ("out"). By using Raman differenc e spectroscopy, we previously have identified characteristic flavin marker bands for the in and out conformers in the solution phase. Now we show that the flavin Raman bands can be used to probe these conformational states in crystals, permitting a comparison between solution and crystal environment s. The in or out marker bands are similar for the respective conformers in the crystal and in solution; however, significant differences do exist, sho wing that the environments for the flavin's isoalloxazine ring are not iden tical in the two phases. Moreover, the Raman-band widths of the flavin mode s are narrower for both in and out conformers in the crystals, indicating t hat the flavin exists in a more limited range of closely related conformati onal states in the crystal than in solution. In general, the ability to com pare detailed Raman data for complexes in crystals and solution provides a means of bridging crystallographic and solution studies.