Sample saving techniques for solution X-ray scattering studies of biological macromolecules
Katz, Andrea Michelle
Small-angle X-ray scattering (SAXS) provides structural information about biomolecules in solution. The resulting insight increases our understanding of biological processes and can aid in structure-based drug design. However, SAXS experiments require tens of microliters of sample at mg/mL concentrations, making the technique unsuitable for molecules that cannot be produced in large quantity. This dissertation introduces three new techniques for sample efficient SAXS experiments. The first details the fabrication and use of fixed path length sample cells for cryoSAXS experiments, as well as their challenges. The cells have rigid walls with low scatter and high X-ray transmission, and allow SAXS measurements from less than two microliters of sample. Although fractures in the vitrified samples produce irreproducible scatter at the lowest angles, the technique is robust and applicable to molecules with maximum dimension less than ~160 Å. The second method implements a coaxial, continuous flow diffusive mixer for low sample volume time-resolved SAXS on the 10 ms - 3 s timescale. The mixer’s geometry allows the use of a larger, higher flux beam than is compatible with most continuous flow SAXS mixers, shortening the acquisition time and reducing sample consumption. A custom beamline setup reduces background scatter to further improve data quality. Each measurement uses less sample than a conventional static SAXS experiment. A study of RNA folding initiated by Mg2+ is presented as an example of the utility of this technique. The final method employs a chaotic advection mixer for time-resolved SAXS measurements on slowly diffusing systems. This device incorporates a miniaturized static mixer to efficiently mix two liquids in the laminar flow regime. An improved beamline setup delivers a higher flux beam than can be used with the diffusive mixer, minimizing acquisition time and sample consumption. This mixer uses an order of magnitude less sample than turbulent flow SAXS mixers, and about twice the sample needed for a conventional static SAXS measurement. Time-resolved SAXS measurements of the binding of trypsin and aprotinin, a well-studied protein pair, are presented as a proof of principle.
cryoSAXS; small angle x-ray scattering; sample saving; Time-resolved; SAXS; microfluidic mixer; Biophysics; Applied physics
Craighead, Harold G.; Gruner, Sol Michael
Ph. D., Applied Physics
Doctor of Philosophy
dissertation or thesis