Abstract
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.
Highlights
X-ray footprinting (XF) mediated by the chemical activity of hydroxyl radicals was first demonstrated as a valuable tool for examining the structure and dynamics of biological macromolecules by studies of Brenowitz, Woodson, and Chance elucidating the folding mechanism of the Tetrahymena ribozyme over 20 years ago (Sclavi et al, 1997, 1998)
11–13 modified residues were observed for cytochrome c, while, for experiments conducted on XFP, 11 and 14 modified residues (Fig. S1) were identified for cytochrome c exposed using capillary flow and multi-sample holder (MSH), respectively
capillary flow (CF) and MSH benchmarking experiments both resulted in reproducible, radiolytic labelling of the sample, and all dose-response plots for both datasets are available in the supporting information
Summary
X-ray footprinting (XF) mediated by the chemical activity of hydroxyl radicals was first demonstrated as a valuable tool for examining the structure and dynamics of biological macromolecules by studies of Brenowitz, Woodson, and Chance elucidating the folding mechanism of the Tetrahymena ribozyme over 20 years ago (Sclavi et al, 1997, 1998). XFP will continue the footprinting program established by X28C, and significant effort is underway to develop new methodologies to study highly scavenging biological samples that have proven difficult to probe using previously available beamlines Complex biological systems such as macromolecular complexes, membrane proteins, and live cells often require high concentrations of various hydroxyl-radical-quenching reagents to preserve sample integrity, and require greater radiation doses to overcome the effect of scavenging secondary reactions and to ensure sufficient radiolytic labelling of the protein sample (Gupta et al, 2007). Degradation of the sample leads to chemical noise in LC-MS spectra, which makes it more difficult to accurately identify and quantify the observed modified and unmodified peptides These issues can be partially circumvented by increasing the flux density of the beam, which increases the steady-state concentration of hydroxyl radicals without any need to extend exposure time (Gupta, Celestre et al, 2014). We anticipate the improved design of XFP will enable us to perform XF experiments that will allow significant advances in the field of hydroxyl radical footprinting of macromolecules
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