Recent Advances in X-Ray Hydroxyl Radical Footprinting at the Advanced Light Source Synchrotron

Abstract

Background: Synchrotron hydroxyl radical footprinting is a relatively new structural method used to investigate structural features and conformational changes of nucleic acids and proteins in the solution state. It was originally developed at the National Synchrotron Light Source at Brookhaven National Laboratory in the late nineties, and more recently, has been established at the Advanced Light Source at Lawrence Berkeley National Laboratory. The instrumentation for this method is an active area of development, and includes methods to increase dose to the samples while implementing high-throughput sample delivery methods.

Conclusion: Improving instrumentation to irradiate biological samples in real time using a sample droplet generator and inline fluorescence monitoring to rapidly determine dose response curves for samples will significantly increase the range of biological problems that can be investigated using synchrotron hydroxyl radical footprinting.

Keywords: Advanced Light Source; Synchrotron; beamline; footprinting; hydroxyl radical footprinting; radiolysis; radiolytic labeling; x-ray..

Figure 1.

Black line: Calculated absorption spectrum assuming a water-based sample in a full range X-ray beam from an ALS bend magnet at 500 mA ring current and 1.9 GeV ring energy, a 150 μm sample thickness, 1 cm of air transmission, and a 125 μm beryllium window. Gray line: same calculation including an 80 μm glass window. Calculations performed using data from the Center for X-ray Optics Web application (cxro.lbl.gov).

Figure 2.

Dose response curves for a 40 kDa protein at a focused (left) vs unfocused (right) white-light bend magnet beamline source at the ALS. Top: comparison of exposure times necessary for the same amount of modification. Solid lines (red and black) represent pseudo unimolecular fit using full and first 3 data points respectively. Solid blue line represents a fit to y=y0+Aexp(−kt). Bottom: Total Ion Chromatogram (TIC) achieved for 0 and maximum dose. More damage was observed for the N-terminal portion of the protein using 50 msec exposure as opposed to 0.6 msecs.

Figure 3.

Left: Effect of X-ray dose on Alexa488 fluorescence quenching between ADE (150 μm droplet diameter, 1 ms exposure), microfluidic flow (200 μm ID, 1 ms exposure), and simple droplet falling under gravity (drop volume 2.5 microliter, point of acceleration 3.2 mm before the beam intersection, 40 ms exposure). Experiment was carried out at ALS 3.2.1. Error represents standard deviation from the mean from 6/7 experimental repeats. ADE ejection showed a 2-fold increase in dose relative to capillary flow, while falling drops showed a 4-fold increase. Right: Theoretical prediction to show the range of drop velocity that will be most useful for HRF. The dots represent the optimum range of velocities for a given diameter of the droplet, assuming that the FWHM of the beam is equal to the drop diameter. Note that under this condition a large drop will have lower dose and will require slower speed to get adequate modification. The ideal range of drop diameter (FWHM of the focused beam) and drop velocities for HRF are shown within the box.

Figure 4.

High-speed camera images of falling drops with various concentrations of Alexa488 dye (left) and manual colorimetry measurements of the drops (right).

Figure 5.

Calculated spot size at sample at ALS beamline 3.3.1 assuming NSLS X18A mirror at 2:1 geometry. Ideal focus (left) is 125 × 91 μm, while focus with estimated slope errors (right) is 500 × 90 μm.