X-ray Footprinting at Beamline X28C: A National Resource for Studying Macromolecular Structure and Dynamics

Journal Article · Thu Jan 01 00:00:00 EST 2009 · Biophysical Journal

DOI:https://doi.org/10.1016/j.bpj.2008.12.243· OSTI ID:980684

D'Mello, R; Gupta, S; Bohen, J; Abel, D; Toomey, J; Sullivan, M; Chance, M

X-ray footprinting employs intense X-rays produced by synchrotron radiation to generate hydroxyl radicals in solution on microseconds-milliseconds timescales. These hydroxyls radicals undergo stable reaction with solvent accessible sites of macromolecule and produce covalent modifications, which are appropriate to probing macromolecule dynamics under physiological condition. For nucleic acids, one analyzes the pattern of fragments after X-ray exposure by gel electrophoresis; the protected sections that are not cleaved yield a 'footprint'. For proteins, the exposed samples are digested with proteases and analyzed by liquid chromatography- and tandem-mass spectrometry to determine the extent and sites of modification. The data provide detailed structural information to map tertiary contacts of macromolecular interactions, which can subsequently be used as constraints for molecular modeling to generate high-resolution structures. This method is unique in providing 'local' structural information in solution for gaining insight into dynamic processes involving, large RNA-protein and protein-protein assemblies on biologically relevant timescales. The method also can uniquely probe the 'local' structure of large complexes poised at equilibrium for functional states of interest, and has been extended to in vivo studies. Beamline X28C is located at the National Synchrotron Light Source of Brookhaven National Laboratory. 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 and Bioinformatics of Case Western Reserve University. The facility supports both onsite and offsite user access. Beam time is allocated online through peer reviewed user proposal system. Examples of recent research projects are provided.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 980684
• Report Number(s): BNL-93602-2010-JA; BIOJAU; TRN: US1005544
• Journal Information: Biophysical Journal, Vol. 96, Issue 3; ISSN 0006-3495
• Country of Publication: United States
• Language: English

Related Subjects

43 PARTICLE ACCELERATORS
BEAMS
BNL
CHROMATOGRAPHY
COMPLEXES
DYNAMICS
ELECTROPHORESIS
EQUILIBRIUM
FUNCTIONALS
GELS
HYDROXYL RADICALS
IN VIVO
NSLS
NUCLEIC ACIDS
PROTEINS
RADICALS
RESERVES
RESOURCES
SIMULATION
SOLUTIONS
SOLVENTS
SPECTROSCOPY
SYNCHROTRON RADIATION
SYNCHROTRONS
national synchrotron light source