NOVEL SOLUTION SAXS GEOMETRY FOR HIGH THROUGHPUT PROTEIN STRUCTURE CHARACTERIZAT

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The development of a novel data collection method for high throughput solution SAXS studies has been initiated, and uses sample aliquots that are suspended in the air as a droplet at the tip of a syringe needle. This new geometry eliminates the windows that are required for conventional sample aliquot containers, minimizing the background scattering level and eliminating the need for cleaning sample cells between each measurement. The possible use of smaller sample volumes than those required for the standard sample containers would allow larger number of sample screening conditions, and characterization of proteins that are very hard to express or purify. Another benefit is the potential elimination of air bubbles, which can appear in the sample aliquot within a capillary cell during sample delivery or oscillation. We have tested 5 and 3 microliter sample aliquot sizes to evaluate the feasibility of this approach for lysozyme solution x-ray scattering. The initial effort focused on evaluating the effects of spherical droplet shape on scattering data and validity of intensity scaling using the transmitted beam intensity. The Blu-Ice controlled Hamilton dispenser allowed the reproducible measurement of small sample aliquots of identical volume. Both water and lysozyme scattering profiles recorded in droplets are essentially identical to those obtained in a capillary cell in the Q range 0.015 - 0.6 A-1, demonstrating that the droplet shape effect is not a major concern and that the standard intensity scaling method works satisfactorily well. However, gradual time-dependent decay in scattering intensities was observed in both cases at the % level over several minutes, likely caused by solvent (water) evaporation which shortens the x-ray path length. Future studies will involve refrigerating the syringe needle to minimize this effect.