SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline

Abstract

Synchrotron X-ray footprinting complements the techniques commonly used to define the structure of molecules such as crystallography, small-angle X-ray scattering and nuclear magnetic resonance. It is remarkably useful in probing the structure and interactions of proteins with lipids, nucleic acids or with other proteins in solution, often better reflecting the in vivo state dynamics. To date, most X-ray footprinting studies have been carried out at the National Synchrotron Light Source, USA, and at the European Synchrotron Radiation Facility in Grenoble, France. This work presents X-ray footprinting of biomolecules performed for the first time at the X-ray Metrology beamline at the SOLEIL synchrotron radiation source. The installation at this beamline of a stopped-flow apparatus for sample delivery, an irradiation capillary and an automatic sample collector enabled the X-ray footprinting study of the structure of the soluble protein factor H (FH) from the human complement system as well as of the lipid-associated hydrophobic protein S3 oleosin from plant seed. Mass spectrometry analysis showed that the structural integrity of both proteins was not affected by the short exposition to the oxygen radicals produced during the irradiation. Irradiated molecules were subsequently analysed using high-resolution mass spectrometry to identify and locate oxidized amino acids. Moreover, the analyses of FH in its free state and in complex with complement C3b protein have allowed us to create a map of reactive solvent-exposed residues on the surface of FH and to observe the changes in oxidation of FH residues upon C3b binding. Studies of the solvent accessibility of the S3 oleosin show that X-ray footprinting offers also a unique approach to studying the structure of proteins embedded within membranes or lipid bodies. All the biomolecular applications reported herein demonstrate that the Metrology beamline at SOLEIL can be successfully used for synchrotron X-ray footprinting of biomolecules.

The XFP (17-BM) beamline for X-ray footprinting at NSLS-II

“…The maximum rate (67000 s À1 ) measured for Alexa 488 fluorophore exposed using high-speed capillary flow at XFP was 229-fold greater than the rate published for SOLEIL, 33fold higher than the highest rate published for NSLS X28C, and 4.9-fold greater than the maximum rate published for beamline 5.3.1 at ALS (Table S1 of the supporting information) (Gupta et al, 2007Baud et al, 2017). It should be noted that this value was attained using a 152 mm aluminium (Al) attenuator, while the analogous measurements at other beamlines were conducted in air or vacuum.…”

Hydroxyl Radical Protein Footprinting: A Mass Spectrometry-Based Structural Method for Studying the Higher Order Structure of Proteins

“…55 In this study, the folding pattern of a 16S rRNA structure was identified in E. coli. As access to synchrotrons is limited, recent efforts have focused on evaluating the ability to perform footprinting experiments at existing synchrotrons 56,57 and establishing formal protocols, 5 making the technique more available. Most recently, the XFP (17-BM) beamline for X-ray footprinting at NSLS-II came online and features unparalleled flux density with the capability of reaching 4.6 × 10 17 photons.…”

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X-ray Beamline

“…HRPF is an umbrella term for covalent labeling approaches employing •OH adducts, where the •OH can be generated by a variety of approaches: radiolysis of water by X-rays, γ-rays, electron beams, electric discharges or plasma sources; decomposition of hydrogen peroxide (H 2 O 2 ) using transition metal-based Fenton chemistry; and photolysis of H 2 O 2 using lasers (FPOP) or a high-pressure flash oxidation lamp. − Among these •OH sources, high-intensity X-ray synchrotron beamlines played an essential role in the beginning of the HRPF field and continue to be important resources for HRPF method development and applications. − X-ray beamlines deliver a measurable, reproducible, and controlled •OH dose on microsecond to millisecond (and longer) time scales. The availability of a high •OH dose at synchrotron beamlines for the structural biology community has allowed studies of complex and highly scavenging systems such as virus assembly, neurodegenerative diseases, epitope mapping, and time-resolved macromolecular dynamics. − Until now, MS analysis steps for X-ray irradiated samples have been carried out at geographically distant sites from the synchrotron, resulting in a slow feedback loop between sample chemistry optimization, beamline experiments, and data analysis .…”