NOVEL SOLUTION SAXS GEOMETRY FOR HIGH THROUGHPUT PROTEIN STRUCTURE CHARACTERIZAT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 已经开始开发一种用于高通量溶液SAXS研究的新的数据收集方法，并使用悬浮在空气中的样品等分作为注射器针头尖端的液滴。这种新的几何形状消除了传统样品等分容器所需的窗口，最大限度地减少了背景散射水平，并消除了每次测量之间清洗样品池的需要。可能使用比标准样品容器所需的样品体积更小的样品体积，将允许更多数量的样品筛选条件，并对很难表达或纯化的蛋白质进行表征。另一个好处是有可能消除气泡，气泡可能在样品输送或振荡过程中出现在毛细管池内的样品等分中。我们测试了5微升和3微升样品等分尺寸，以评估该方法用于溶菌酶溶液X射线散射的可行性。最初的工作集中在评估球形液滴形状对散射数据的影响以及利用透射束强度进行强度定标的有效性。Blu-Ice控制的哈密尔顿分配器允许对相同体积的小样本等分进行可重复测量。在液滴中记录的水和溶菌酶散射曲线在0.015-0.6A-1范围内与在毛细管池中获得的基本相同，表明液滴形状效应不是主要问题，标准强度定标方法工作得令人满意。然而，在这两种情况下，在几分钟内都观察到了散射强度随时间的逐渐衰减，这可能是由于溶剂(水)蒸发缩短了x射线路径长度造成的。未来的研究将包括冷藏注射器针头，以将这种影响降至最低。

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.