X-ray Footprinting
Structure determination through probing solvent accessible regions of biomolecules

Left: With the method of X-ray footprinting, an X-ray beam interacts with water in solution to produce hydroxyl radicals, which covalently modify proteins in solvent-accessible regions; these modifications are detected using standard liquid chromatography mass spectrometry (LC-MS). Right: Highlights of the types of structural information that can be obtained using the method, from dynamics of G-protein-coupled receptors (GPCR) activation, to prion surface features, to how proteins fit together in cellular structures.[Image Credits: (1) Lawrence Berkeley National Laboratory; (2) Reprinted from Du, Y., et al. 2019. Assembly of a GPCR-G Protein Complex, Cell 177(5), 1232-42, with permission from Elsevier; (3) Reprinted under a Creative Commons Attribution 4.0 International License (CC BY 4.0) from Sommer, M., et al. 2019. DOI: 10.1104/pp.18.01190; (4) Lawrence Berkeley National Laboratory; (5) Case Western Reserve University.]
Conceptual overview available here: https://youtu.be/wnnbICWUTjA.
Key Features of X-ray Footprinting
- Data obtained is used to construct a “water map,” revealing locations of water molecules at the time of X-ray exposure
- Typical X-ray exposure time on the order of microseconds or milliseconds
- Yields protein conformation or protein-protein interaction regions as a function of time
- Oxidative modifications are permanent and preserved post-exposure so samples can be stored for later analysis by liquid chromatography mass spectrometry (LC-MS) or sequencing, as appropriate to the sample type
BER Researchers Use X-ray Footprinting to Study:
- Evolution of protein structure and protein-protein interaction in solution under both steady-state or time-resolved conditions, including in response to outside stressors
- Protein or nucleic acid folding or unfolding mechanisms
- Protein-nucleic acid interactions
- Develop structural understanding of intrinsically disordered proteins that are challenging for standard methods
- Examination of protein or nucleic acids structure in intact viruses or from brain tissue
- Small molecule interactions with proteins, such as drug binding to targets or binding of proteins to inorganic structures
Sample Considerations
- 5-10 micromolar concentration
- 5-200 microliters sample volumes depending on the experiment
- Sample buffers should be minimally scavenging (e.g., no glycerol)
- 96-well plate or liquid jet/capillary sample delivery options
- On-site or remote beamline access; LC-MS on demand
X-ray Footprinting Beamlines at DOE User Facilities
- X-ray Footprinting of Biological Materials at NSLS-II, BNL (17-BM XFP)
- X-ray Footprinting at the ALS at ALS, LBNL (3.3.1)
References
- Du, Y., et al. 2019. “Assembly of a GPCR-G Protein Complex,” Cell 177(5), 1232-42. DOI: 10.1016/j.cell.2019.04.022.
- Sommer, M., et al. 2019. “Heterohexamers Formed by CcmK3 and CcmK4 Increase the Complexity of Beta Carboxysome Shells,” Plant Physiology 179(1), 156-67. DOI: 10.1104/pp.18.01190.
- Schoof, M., et al. 2020. “An Ultrapotent Synthetic Nanobody Neutralizes SARS-CoV-2 by Stabilizing Inactive Spike,” Science 370(6523), 1473-79. DOI: 10.1126/science.abe3255.
- Kamali-Jamil, R., et al. 2021. “The Ultrastructure of Infectious L-type Bovine Spongiform Encephalopathy Prions Constrains Molecular Models,” PLoS Pathogens 17(6), e1009628. DOI: 10.1371/journal.ppat.1009628.