The Beamline X28C of the Center for Synchrotron Biosciences: a National Resource for Biomolecular Structure and Dynamics Experiments Using Synchrotron Footprinting

Abstract

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 Hybrid Structural Approach to Analyze Ligand Binding by the Serotonin Type 4 Receptor (5-HT4)

“…Samples from three different preparations were exposed to broadband focused x-rays at Beamline X28C of the National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY (19,20). X-ray beam parameters were optimized by using the standard fluorophore assay that monitors the loss of intensity of an Alexa 488 fluorophore (Invitrogen) to reveal the effective hydroxyl radical concentration under selected experimental conditions (19).…”

“…Radiolytic Protein Footprinting Analyses-The rate of oxidation of a residue side chain depends on both its reactivity with hydroxyl radicals and its accessibility to water from which hydroxyl radicals are formed during radiolysis (19,22,23,32). Forty four residues within the 5HT4R sequence were found modified by oxidation, plus three residues were modified in the C-terminal tag in the 41 unique peptides detected by MS.…”

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth

“…Radiolysis experiments were performed at beamline X28X of the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, New York, USA) and at beamline 5.3.1 of the Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley, California, USA). The x-ray beam parameters were optimized by using Alexa Fluor 488 fluorophore assay (41). All samples were exposed for 0 to 20 milliseconds (X28X) and for 0 to 800 μs (5.3.1) at ambient temperature and immediately quenched with methionine amide at a 10 mM final concentration to prevent secondary oxidation (42).…”

Structural Characterization of HIV gp41 with the Membrane-proximal External Region

“…Three sets of samples (50-l injection volume) were exposed to a mirror-focused (31) synchrotron x-ray beam (5.5-milliradian angle, focus value of 6) at the X28C beamline of the National Synchrotron Light Source at Brookhaven National Laboratory for 0 -20 ms. The exposure time of the samples was controlled via flow rate through the flow cell in the KinTek (Austin, TX) stopped-flow apparatus (30). Oxidation was quenched by the flow of sample from the KinTek into the collection vessel containing methionine amide (final concentration of 10 mM).…”

“…Diluted samples were kept at 4°C prior to the experiments and used within 12 h of thawing. Exposure conditions were predetermined by following the dose-dependent degradation of the fluorescent compound Alexa 488 (Invitrogen) in the presence of the sample in the sample buffer (30). Three sets of samples (50-l injection volume) were exposed to a mirror-focused (31) synchrotron x-ray beam (5.5-milliradian angle, focus value of 6) at the X28C beamline of the National Synchrotron Light Source at Brookhaven National Laboratory for 0 -20 ms.…”