Combining on-line spectroscopy with synchrotron and X-ray free electron laser crystallography

The mechanism of enzymatic reactions and the associated kinetics are commonly studied using spectroscopic methods in solution, while the structures in the crystalline state are determined with X-ray crystallography, most often using cryo-cooled crystals. The structures of intermediate states are, when possible, determined from crystals that are generated using freeze-quench techniques at different times of the enzymatic reaction. However, most such studies do not use spectroscopy and crystallography simultaneously online, and it is difficult to correlate the structures with the spectroscopic information independently obtained using solution samples. With the advent of X-ray free-electron lasers (XFELs) and the establishment of serial crystallography (SX) over the last five years, it has become possible to determine the structure of intermediate states generated in situ. Although such X-ray crystallographic studies provide detailed structural information of enzymes and their active sites, such data alone are insufficient to characterize the reaction kinetics, ligand formation, and oxidation state of the metals in the catalytic site among other key features of protein dynamics. In contrast, different modes of spectroscopy, while unable to generate direct three-dimensional structural information, are capable of clearly identifying such elements of protein dynamics that cannot be attained through X-ray diffraction (XRD) data. Combining structural studies with simultaneous on-line in situ spectroscopy, therefore, provides a powerful method to characterize the intermediate states independently. The often complex and multiphasic nature of enzyme catalysis and protein dynamics makes it especially important to establish the reaction timescale and population of the intermediate states (which becomes possible using spectroscopy), that are critical to correctly determine the structures of the intermediate species of interest. Crystallographic data of a transient state often consists of a combination of discrete (meta)stable states that can only be deconvolved accurately with knowledge of the composition of such mixtures. This piece of information can be critically assessed by time-resolved spectroscopy methods. Following this need, several recent crystallography studies have been coupled with spectroscopic characterization of protein samples in both solution and crystal forms. Notably, application of in crystallo spectroscopy has been significantly beneficial in determining the enzyme structure, function and reaction kinetics, and mechanism in comparison to those in solution [1, 2, 3, 4].

Another important benefit of combined X-ray crystallography and spectroscopy at ambient temperature is the ability to monitor X-ray-induced modification of the sample. The radiation-induced ‘damage’ process involves generation and migration of radicals that affect both the geometric and the electronic structure and has been one of the major challenges in studying metalloenzymes using synchrotron-based X-ray methods [5, 6, 7]. While this problem can be partly mitigated by collecting data at cryogenic temperatures and by using several crystals for data collection, it still is a serious limitation for room temperature time-resolved studies. With the advent of XFELs [8], the condition has been changed drastically as the highly intense and ultrafast X-ray pulses generated by XFELs are able to probe the sample on a timescale faster than that of the damage process [9, 10, 11]. At ambient temperatures, XFEL is commonly employed in an approach known as serial femtosecond crystallography (SFX) where microcrystals are replaced between each X-ray pulse [12,13]. In this approach, up to hundreds of thousands of crystals are probed to construct a complete diffraction dataset. Combined SFX/spectroscopy experiments make it possible to monitor the integrity of the samples. While larger physical quantities of protein samples are probed in SFX experiments, the ability to closely monitor sample quality such as batch-to-batch homogeneity via simultaneous spectroscopic measurements can improve the quality of these serial experiments.

Given the above described benefits for studies of protein reaction mechanisms and dynamics, as well as the concerns regarding X-ray-induced damage in synchrotron studies and sample quality in serial experiments, different modes of spectroscopy have been combined with the crystallography experiments at both synchrotron and XFEL light sources. Since many enzymes harbor chromophores, characteristic ligand environments, and/or bioinorganic active sites that give rise to distinct spectroscopic signatures, modes of on-line spectroscopy range from infrared to optical to X-ray, realized using multiple approaches for different purposes. Importantly, tandem measurement of spectra and diffraction from the same sample under identical conditions enable spectroscopy to serve as rapid feedback for crystallography.

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