Infrared protein crystallography

Crystallography has become so strongly associated with X-ray diffraction that the two terms are nearly synonymous in the minds of many practicing scientists. However, it is worth recalling that crystallographers were making important contributions to the understanding of matter long before Rontgen's discovery of X-rays in 1897. In the 19th century, for example, crystallographers were leading proponents of the atomic theory of matter, because it allowed successful quantitative explanations of many crystal shapes. Optical crystallography, over a growing frequency range from infrared to X-ray, continues to provide molecular insights. Interest in spectroscopic measurements in the crystalline phase is increasing [1], [2], [3], [4], [5], [6], [7] and [8]. This is largely driven by the desire to establish that the detailed three-dimensional structural models derived from X-ray diffraction on protein crystals actually coincide with the structures of the proteins in solution. In addition to the possibility that intermolecular interactions in the crystalline environment may perturb the structure, there is a growing realization that intense X-radiation from synchrotron sources modifies the molecules under study [9], [10], [11] and [12]. This is especially a concern with regard to accurate structural description of metalloprotein active sites, where the redox activity that is integral to their biological function also renders them more susceptible to structural changes by trapping photoelectrons [13], [14], [15], [16], [17], [18] and [19]. Single crystal spectroscopy can also identify protein intermediate states in kinetic crystallography, which aims at their generation, trapping, and structural characterization [20]. Unique features of the crystalline environment, most notably the high degree of molecular orientation, also create opportunities to obtain spectroscopic information that is not available from measurements on solutions. Crystalline spectroscopy has emphasized electronic spectroscopy. However, vibrational spectroscopy provides a number of advantages because of its higher information content. This article focuses on infrared measurements on protein crystals—experimental methodology and applications.