Development of a High Throughput X-Ray Footprinting Facility at the Advanced Light Source to Study the Structure and Dynamics of Complex Biological Macromolecules

Abstract

Radiolytic footprinting is an increasingly popular method for structural elucidation of macromolecules in the solution state. X-ray footprinting in particular has developed in the past decade into a nearly routine technique and had been applied to a diverse range of biological systems, yielding unique structural insights impossible to obtain using the more standard structural methods of crystallography, NMR, electron microscopy and small angle scattering. The technique was pioneered at the National Synchrotron Light Source (NSLS) over the past decade; now with the user community continuing to grow and the anticipated closure of this facility, it is imperative that x-ray footprinting continue to be developed. Towards this end we are building a new footprinting beamline at the Advanced Light Source (ALS) synchrotron which will not only support the NSLS users during the commissioning of the NSLS-II, but will provide unique capabilities for the continued development of the method. In particular, our preliminary data show that flux densities produced by an ALS focused white-light bend magnet beamline are high enough to allow microsecond exposures yielding sufficient modification to conduct footprinting experiments. In addition, in-house mass spectrometry availability and expertise will allow the installation of a mass spectrometer instrumentation directly at the beamline. Combined with commercially available tools for automated sample handling, rapid sample digestion and data processing methods, this will allow rapid screening of a large number of samples and will be especially important for samples sensitive to degradation. The high-throughput methodology and microsecond regime made available at the ALS beamline will be highly complementary to the planned NSLS-II footprinting beamline, and both resources will be necessary to accommodate the growing national user community.