Synchrotron X-ray footprinting is a valuable method to observe the structure and dynamics of biological macromolecules and their interactions with each other, as well as with smaller molecules such as drug candidates, in the native solution state. Intense, broadband ionizing radiation produced by a synchrotron activate water molecules in solution, generating hydroxyl radicals that can then react with components of the solution, such as biomolecules, on microsecond-millisecond timescales. This time-frame is well-suited to probe macromolecule dynamics while minimizing sample perturbation. Hydroxyl radicals generated in this fashion readily attack solvent-accessible protein side chains, ultimately leading to a covalent modification of the side chain. In the case of nucleic acids, the radicals cleave the phosphodiester backbone, again in a solvent accessible fashion. For nucleic acids, one typically analyzes the pattern of fragments after X-ray exposure by gel electrophoresis, and the protected sections that are not cleaved yield a footprint (see a nucleic acid FP workflow diagram). For proteins, a bottoms-up proteomic workflow is normally adopted, using protease digestion and analysis via modern LC-coupled electro-/nano-spray mass spectrometry approaches to generate a peptide coverage map and identify specific sites within each peptide that are covalently modified (see a protein FP workflow diagram). The labeling or cleavage readout provides detailed structural information, down to single-nucleotide or single side-chain levels, that is used to map both structure and regions of macromolecular interaction.
Classical footprinting approaches involve pairwise comparisons between two states of a biomolecule. That is, one measures the footprint of the native biomolecule and then introduces a ligand or other biomolecule binding partner and compares the two readouts for changes, as these binding/folding events can lead to changes in the solvent accessibility of different regions of the target biomolecule, thus altering the rate and extent of reaction with hydroxyl radicals. These changes in reactivity can be correlated to, for instance, protection of a region by binding of a ligand, a change in the folded conformation, or an allosteric change. In and of itself, XF generally cannot unambiguously discern between these various possibilities. However, structure hypotheses can be validated by combining XF data with parameters from other structural probes such as protein crystallography or NMR (atomic-level structures of proteins or nucleic acids), small angle X-ray scattering (global structural envelopes), Cryo-EM (both global and local structure), as well as classical biochemical methods including mutagenesis. In addition, more recent methodological developments, such as iSPOT, hold promise for using footprinting as an absolute structure prediction tool, particularly for problems such as intrinsically disordered proteins, where crystallography or cryo-EM are less amenable.
Research Programs and Equipment: NSLS-II XFP
The CSB has developed and constructed a beamline for X-ray footprinting, XFP (X-ray Footprinting of Biological Materials), located at the National Synchrotron Light Source II (NSLS-II) at Brookhaven National Laboratory (BNL) in Upton, NY. In addition to operating the beamline itself as a partner with the NSLS-II Structural Biology Program, the CSB’s XFP beamline team designs and implements state-of-the-art instrumentation and sample handling equipment for synchrotron footprinting.
Currently, most X-ray footprinting experiments are performed using a high-throughput 96-well device (5 µL sample in 200 µL PCR tubes, strips, or plates) for steady-state experiments from -35 °C to +37 °C (exposure times >10 ms). This device can accommodate specimens requiring biosafety level 2 (BSL-2) controls in risk groups 1, 2, and 3. Samples requiring very high hydroxyl radical doses on short time scales, such as membrane proteins or large multicomponent complexes, can be examined using capillary flow methods (200 or 530 µm ID capillaries), with a nominal sample volume of 50-100 µL per exposure and exposure times ranging from 75 µs to 2 ms using a syringe pump. This device can also be coupled to a continuous flow sample pump, enabling, for example, live cell culture experiments with an in-line incubator and fraction collector. Sample 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.