Nonlinear Optical Imaging for Guiding Protein Structure Determination

 描述（由申请人提供）：通过X射线衍射确定的高分辨率蛋白质结构需要生产高质量、有序的晶体。这种晶体的产生通常涉及初始结晶条件的确定，随后进行优化以产生良好有序的晶体，以及随后的X射线衍射，最常见的是在同步加速器设施中进行。该管道中的每个步骤通常需要数周或数月来评估迭代优化的成功或失败，受限于蛋白质成核和生长的缓慢动力学的组合，以产生大的有序单晶， 以及对用于评估晶体质量和执行数据收集的同步加速器设施的有限访问。 我们建议使用非线性光学成像，以迅速通知最后三个关键的实验步骤，初始晶体形成，优化晶体质量之前，以acetactin，和同步加速器X射线衍射分析。对于第一个目标，提出了用“SHG-发色团”插入蛋白质晶体以增强通过SHG可检测的晶体的范围和尺寸，概念验证测量表明可以实现1000倍的增强因子。在现场评估晶体质量提出了快速优化的基础上使用偏振相关的SHG成像识别多域，孪生，和高度镶嵌晶体。最后，基于同步加速器XRD与多模式共焦反射率、明场透射率、双折射、双光子激发紫外荧光（TPE-UVF）的集成，提出了实现微米和纳米晶体簇射快速连续结晶学的仪器。和UV-SHG，所有这些都以高达视频速率同时获取并具有完美的图像配准。使用这种组合测量套件，我们的目标是实现直径为1- 5 mm的晶体的常规和可靠检测，以实现1 mm准直源的衍射测量。 拟议的努力建立在前一个NIH支持初始周期奠定的基础上。SHG和TPE-UVF显微镜在我们之前的支持期间首次提出，现在已经在晶体学领域建立并广泛使用。以前的支持直接促成了35篇同行评审期刊文章，66篇演讲和3项已颁发的专利。我们的技术已获得许可，商业SHG/TPE-UVF显微镜现在可用于蛋白质晶体筛选。此外，定制的第一代非线性光学成像仪器已被集成到阿贡国家实验室的大分子晶体学光束线中。

 DESCRIPTION (provided by applicant): High-resolution protein structures determined by X-ray diffraction require the production of high-quality, well-ordered crystals. The generation of such crystals typically involves the identification of initial crystallization conditions, followedby optimization to produce well-ordered crystals, and subsequent X-ray diffraction, most commonly performed at synchrotron facilities. Each step in this pipeline can typically require weeks or months to assess success or failure in iterative optimizations, limited by a combination of slow kinetics for protein nucleation and growth in order to generate large well-ordered single crystals, and limited access to synchrotron facilities for assessing crystal quality and performing data collection. We propose the use of nonlinear optical imaging to rapidly inform the final three key experimental steps of initial crystal formation, optimization of crystal quality prior to diffractin, and synchrotron X-ray diffraction analysis. For the first goal, intercalation of protein crystals wth "SHG-phores" is proposed for enhancing the range and sizes of crystals detectable by SHG, with proof-of-concept measurements suggesting enhancement factors of 1000-fold are achievable. In situ assessment of crystal quality is proposed for rapid optimization based the use of polarization-dependent SHG imaging for identification of multi-domain, twinned, and highly mosaic crystals. Finally, instrumentation to enable rapid serial crystallography of micro- and nano-crystalline showers are proposed based on integration of synchrotron XRD with multi-modal confocal reflectance, brightfield transmittance, birefringence, two-photon excited ultraviolet fluorescence (TPE-UVF), and UV-SHG, all acquired simultaneously at up to video rate and with perfect image registry. Using this combined measurement suite, we aim to enable routine and confident detection of crystals 1-5mm in diameter to enable diffraction measurements with a 1 mm collimated source. The proposed efforts build on a foundation laid during the previous initial cycle of NIH support. SHG and TPE-UVF microscopy first proposed in our previous period of support are now established and widely used methods within the crystallography community. Previous support directly contributed to 35 peer-reviewed journal articles, 66 presentations, and 3 issued patents. Our technology has been licensed, and a commercial SHG/TPE-UVF microscope is now available for protein crystal screening. In addition, a custom first-generation nonlinear optical imaging instrument has been integrated into a macromolecular crystallography beamline at Argonne National Laboratory.