Structural mapping of proteins and nucleic acids with high resolution in solution is of critical importance for understanding their biological function. A wide range of footprinting technologies have been developed over the last ten years to address this need. Beamline X28C, a white-beam X-ray source at the National Synchrotron Light Source of Brookhaven National Laboratory, functions as a platform for synchrotron footprinting research and further technology development in this growing field. An expanding set of user groups utilize this national resource funded by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health. The facility is operated by the Center for Synchrotron Biosciences and the Center for Proteomics of Case Western Reserve University. The facility includes instrumentation suitable for conducting both steady-state and millisecond time-resolved footprinting experiments based on the production of hydroxyl radicals by X-rays. Footprinting studies of nucleic acids are routinely conducted with X-ray exposures of tens of milliseconds, which include studies of nucleic acid folding and their interactions with proteins. This technology can also be used to study protein structure and dynamics in solution as well as protein-protein interactions in large macromolecular complexes. This article provides an overview of the X28C beamline technology and defines protocols for its adoption at other synchrotron facilities. Lastly, several examples of published results provide illustrations of the kinds of experiments likely to be successful using these approaches.

A synchrotron infrared (IR) beamline, U2B, dedicated to the biomedical and biological sciences has been constructed and is in operation at the National Synchrotron Light Source (NSLS) of Brookhaven National Laboratory. The facility is operated by the Center for Synchrotron Biosciences of the Albert Einstein College of Medicine in cooperation with the NSLS. Owing to the broadband nature of the synchrotron beam with brightness 1000 times that of conventional sources, Fourier transform IR spectroscopy experiments are feasible on diffraction-limited sample areas at high signal-to-noise ratios and with relatively short data-acquisition times. A number of synchrotron IR microscopy experiments that have been performed in the mid-IR spectral range (500-5000 cm(-1)) are summarized, including time-resolved protein-folding studies in the microsecond time regime, IR imaging of neurons, bone and other biological tissues, as well as imaging of samples of interest in the chemical and environmental sciences. Owing to the high flux output of this beamline in the far-IR region (50-500 cm(-1)), investigations of hydrogen bonding and dynamic molecular motions of biomolecules have been carried out from 10 to 300 K using a custom-made cryostat and an evacuated box. This facility is intended as an international resource for biological IR spectroscopy fully available to outside users based on competitive proposal.

High-throughput X-ray absorption spectroscopy was used to measure transition metal content based on quantitative detection of X-ray fluorescence signals for 3879 purified proteins from several hundred different protein families generated by the New York SGX Research Center for Structural Genomics. Approximately 9% of the proteins analyzed showed the presence of transition metal atoms (Zn, Cu, Ni, Co, Fe, or Mn) in stoichiometric amounts. The method is highly automated and highly reliable based on comparison of the results to crystal structure data derived from the same protein set. To leverage the experimental metalloprotein annotations, we used a sequence-based de novo prediction method, MetalDetector, to identify Cys and His residues that bind to transition metals for the redundancy reduced subset of 2411 sequences sharing <70% sequence identity and having at least one His or Cys. As the HT-XAS identifies metal type and protein binding, while the bioinformatics analysis identifies metal-binding residues, the results were combined to identify putative metal-binding sites in the proteins and their associated families. We explored the combination of this data with homology models to generate detailed structure models of metal-binding sites for representative proteins. Finally, we used extended X-ray absorption fine structure data from two of the purified Zn metalloproteins to validate predicted metalloprotein binding site structures. This combination of experimental and bioinformatics approaches provides comprehensive active site analysis on the genome scale for metalloproteins as a class, revealing new insights into metalloprotein structure and function.

Hydroxyl-radical mediated synchrotron X-ray footprinting (XF) is a powerful solution-state technique in structural biology for the study of macromolecular structure and dynamics of proteins and nucleic acids, with several synchrotron resources available to serve the XF community worldwide. The XFP (Biological X-ray Footprinting) beamline at the NSLS-II was constructed on a three-pole wiggler source at 17-BM to serve as the premier beamline for performing this technique, providing an unparalleled combination of high flux density broadband beam, flexibility in beam morphology, and sample handling capabilities specifically designed for XF experiments. The details of beamline design, beam measurements, and science commissioning results for a standard protein using the two distinct XFP endstations are presented here. XFP took first light in 2016 and is now available for general user operations through peer-reviewed proposals. Currently, beam sizes from 450 µm × 120 µm to 2.7 mm × 2.7 mm (FWHM) are available, with a flux of 1.6 × 1016 photons s−1 (measured at 325 mA ring current) in a broadband (∼5–16 keV) beam. This flux is expected to rise to 2.5 × 1016 photons s−1 at the full NSLS-II design current of 500 mA, providing an incident power density of >500 W mm−2 at full focus.