Spectroscopic Validation of Crystallographic Structures of a Protein Active Site by Chiroptical Spectroscopy.

Out-of-plane distortions of a cofactor molecule in a protein active site are functionally important, and in photoreceptors, it has been proposed that they are crucial for spectral tuning and energy storage in photocycle intermediates. However, these subtle structural features are often beyond the grasp of structural biology. This issue is strikingly exemplified by photoactive yellow protein: its 14 independently determined crystal structures exhibit considerable differences in the dihedral angles defining the chromophore geometry, even though most of these are at excellent resolution. Here we developed a strategy to verify cofactor distortions in crystal structures by using quantum chemical calculations and chiroptical spectroscopy, particularly Raman optical activity and electronic circular dichroism spectroscopies. Based on this approach, we identify seven crystal structures with the chromophore geometries inconsistent with the experimentally observed data. The strategy implemented here promises to be widely applicable to uncovering cofactor distortions at active sites and to studies of reaction intermediates.