Time-resolved SAXS measurements facilitated by online HPLC buffer exchange

Area of Science:

• Biophysics
• Biochemistry
• Structural Biology

Background:

• Small-angle X-ray scattering (SAXS) is crucial for characterizing biological macromolecules in solution.
• Studying heterogeneous solutions and reactions triggered by species removal presents significant challenges.
• Existing time-resolved SAXS (TR-SAXS) methods often rely on rapid mixing techniques.

Purpose of the Study:

• To present a new TR-SAXS method for studying reactions initiated by the removal of a specific species.
• To demonstrate the applicability of this method using the self-association of long-acting insulin analogues triggered by phenol removal.
• To provide a versatile approach for analyzing dynamic processes in solution.

Main Methods:

• Development of a TR-SAXS method utilizing fast buffer exchange over a desalting column.
• Integration of an online high-performance liquid chromatography (HPLC) system with the SAXS sample cell.
• Stopping the sample flow in the cell to monitor reactions initiated by species removal, such as phenol.

Main Results:

• Successfully obtained TR-SAXS data for a self-association process triggered by phenol removal from long-acting insulin analogues.
• Demonstrated the ability to collect time-series data while varying concentrations.
• Validated a novel method for studying dynamic processes initiated by changes in solution composition.

Conclusions:

• The presented method offers a powerful alternative for TR-SAXS studies, particularly for reactions triggered by species removal.
• This approach expands the scope of TR-SAXS applications to a wider range of biological and chemical systems.
• The technique facilitates detailed structural characterization of dynamic processes in solution.