Synchrotron X-ray Footprinting provides a method of observing the structure and dynamics of biological macromolecules and their interactions in the solution state (in some cases within living cells) at the single nucleotide or single amino acid level. This technique employs the very intense broadband X-ray beams produced by synchrotron radiation to generate hydroxyl radicals in solution for use on microsecond-millisecond timescales. These radicals cleave the phosphodiester backbone of nucleic acids and covalently modify amino acid side chains in proteins. In regions where a molecule is folded, binds a partner, or changes conformation through tertiary or quaternary contacts, the susceptible sites in the macromolecules change their solvent accessibility, and the rate and extent of reaction with radicals is altered. For nucleic acids, one analyzes the pattern of fragments after X-ray exposure by gel electrophoresis (Figure 1); he protected sections that are not cleaved yield a "footprint". For proteins, the exposed samples are digested with proteases and analyzed by mass spectrometry to determine the extent and sites of modification (Figure 2). The data provide detailed structural information (at the single-nucleotide and single side-chain level) that is used to map tertiary structure as well as regions of macromolecular interaction. 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 at higher resolution on biologically relevant timescales and under physiological conditions. Synchrotron based footprinting studies have provided novel insights and approaches for steady state and time-resolved studies of macromolecular dynamics, examining the structure of large macromolecular assemblies, in vivo studies of macromolecules, and even for observing functional water dynamics within the context of these biological structures.
Research Programs and Equipment: NSLS-II XFP
The CSB has developed and constructed a new beamline for X-ray Footprinting, XFP (X-ray Footprinting for in Vitro and in Vivo Structural Studies of Biological Macromolecules), located at the National Synchrotron Light Source II (NSLS-II) at Brookhaven National Laboratory (BNL) in Upton, NY. In addition to constructing the beamline itself, the research team at NSLS-II XFP designs, tests, and implements state-of-the-art instrumentation and sample handling equipment for synchrotron footprinting. The current standard beamline apparatus available for use supports capillary flow experiments (generally 200 µm ID capillary), with a nominal sample volume of 100 µL per exposure and exposure times ranging from 75 microseconds to 2 milliseconds. Equipment currently in advanced stages of commissioning will support high-throughput low-volume (5 µL) steady state sample exposure from -30 °C to 45 °C (exposure times >10 ms), millisecond time scale time-resolved experiments, and multi-pump/syringe pump large volume (up to 60 mL) or continuous flow sample exposure, including live cell culture experiments with in-line incubator and fraction collector. Samples concentrations are generally in the range of 100 nM - 10 µM.
The CSB provides comprehensive beamline user support for research projects conducted by academic, government and industrial institutions. This support includes consultation on experimental design and sample preparation, assistance and training in submission of good beamline proposals, performing the experiments at the beamline, and an introduction to data analysis. Collaborations with beamline staff and/or additional experts in the Center may be available to enhance your program. For more information on the NSLS-II XFP beamline, schedules and contact information please visit the specific XFP webpage.