Development of a microsecond X-ray protein footprinting facility at the Advanced Light Source

Area of Science:

• Structural Biology
• Biophysical Chemistry
• Synchrotron Radiation Science

Background:

• X-ray footprinting (XF) is a vital technique for analyzing macromolecular structure and dynamics in solution.
• The closure of the National Synchrotron Light Source necessitates the development of XF capabilities at alternative synchrotron facilities.
• A new XF program has been established at the Advanced Light Source (ALS) to ensure continued access for researchers.

Purpose of the Study:

• To establish and validate X-ray footprinting capabilities at the Advanced Light Source.
• To demonstrate the accessibility of microsecond timescales for protein footprinting at ALS beamline 5.3.1.
• To enhance radiolytic labeling efficiency and differentiate water molecule environments in protein samples.

Main Methods:

• Utilized white-light bending-magnet beamlines 5.3.1 and 3.2.1 at the Advanced Light Source.
• Employed a focusing mirror and micro-capillary flow cell at beamline 5.3.1 to achieve high flux density.
• Saturated protein samples with nitrous oxide to investigate its effect on radiolytic labeling.

Main Results:

• Demonstrated the feasibility of microsecond time-resolved protein footprinting at ALS beamline 5.3.1.
• Achieved increased radiolytic labeling efficiency by saturating samples with nitrous oxide.
• Successfully distinguished between bound and bulk water molecules within protein samples.

Conclusions:

• The Advanced Light Source is a suitable facility for X-ray footprinting experiments.
• The developed methods enable high-quality structural data acquisition for complex protein samples.
• Microsecond timescale dynamics information can now be obtained using XF at the ALS.