Ultrafast Nonlinear Optical Approaches toward High-Throughput Membrane Protein Na

描述(由申请人提供)：膜蛋白是结构确定中最具挑战性的蛋白质目标之一，也是对人类健康最重要的。X射线光源方面的重大进展，包括X射线自由电子激光(XFEL)衍射的使用、紧密聚焦的同步加速器X射线衍射仪(XFEL)和电子衍射(ED)仪器的改进，已经不可避免地将结构测定所需的晶体尺寸缩小到蛋白质纳米晶区。这些趋势正越来越多地导致蛋白质纳米晶体筛选的主要瓶颈，随着这些新的衍射能力的继续发展，其重要性只会增加。这项拟议工作的主要目标将是开发基于超快激光光源的非线性光学相互作用的测量工具，用于选择性地筛选蛋白质纳米晶体的形成。将开发三种关键的新方法。首先，偏振相关的TPE-UVF将被开发和验证为传统SONICC的补充，用于检测固定的蛋白质晶体，例如在脂类中间相和低温条件下遇到的那些。有序晶体结构域产生不同于聚集体和溶剂化蛋白质的偏振相关的TPE-UVF图案。在第二方面，将发展倍频和TPE-UVF自相关方法，以提高扩散纳米晶的检测下限，目标是从水溶液中产生的2D和3D纳米晶。自相关依赖于分析来自许多纳米晶体的组合弱响应以增强信噪比，同时提供关于平移扩散的信息。最后，提出了利用偏振相关倍频和TPE-UVF选择性检测蛋白质纳米晶转动动力学的非线性光学互相关光谱方法。TPE-UVF的偏振相关性将保持TPE-UVF和SONICC对自由扩散纳米晶体的晶体特异性检测。建议方法的验证将使用聚焦X射线衍射、XFEL衍射和ED相结合进行，其中各种和代表性的膜蛋白纳米晶样品通过与Vadim Cherezov(斯克里普斯)合作提供用于脂类中间相结晶，Petra Fromme(亚利桑那州大学)提供适合XFEL分析的晶体，以及David Stokes(NYU)提供2D纳米晶样品用于ED结构确定。联合SONICC和同步加速器X射线衍射测量将在APS使用能够结合倍频成像和聚焦“微束”X射线衍射的原型进行。虽然目前研究的主要目的是从根本上推进膜蛋白纳米晶检测，但如果拟议的努力被证明是成功的，与Formulatrix的密切合作将有助于降低将这些查询的结果快速转换到高通量商业平台用于常规蛋白质纳米晶检测的障碍。

DESCRIPTION (provided by applicant): Membrane proteins represent among the most challenging protein targets for structure determination and yet also the most important for human health. Major emerging advances in X-ray sources, including the use of X-ray free electron laser (XFEL) diffraction, tightly focused synchrotron XRD, and improvements in electron diffraction (ED) instrumentation have inexorably reduced the crystal sizes required for structure determination into the protein nanocrystal regime. These trends are increasingly leading to a major bottleneck in protein nanocrystal screening, the importance of which will only increase as these new diffraction capabilities continue to advance. The primary objective of the proposed effort will be to develop measurement tools based on nonlinear optical interactions from ultrafast laser sources for selectively screening protein nanocrystal formation. Three key novel approaches will be developed. In the first, polarization-dependent TPE-UVF will be developed and validated as a complement to conventional SONICC for the detection of immobile protein crystals, such as those encountered in lipidic mesophases and under cryogenic conditions. Ordered crystalline domains produce polarization-dependent TPE-UVF patterns distinct from aggregates and solvated proteins. In the second, SHG and TPE-UVF autocorrelation methods will be developed for improving the detection limits of diffusing nanocrystals, targeting 2D and 3D nanocrystals generated from aqueous solutions. Autocorrelation relies on analysis of the combined weak responses from many nanocrystals for signal to noise enhancement, while simultaneously providing information on translational diffusion. Finally, nonlinear optical cross-correlation spectroscopy methods will be advanced using polarization-dependent SHG and TPE-UVF for the selective detection of protein nanocrystal rotational dynamics. The correlations within the polarization-dependence of TPE-UVF will preserve crystal-specific detection by both TPE-UVF and SONICC for freely diffusing nanocrystals. Validation of the proposed approaches will be performed using a combination of focused XRD, XFEL diffraction, and ED, with diverse and representative membrane protein nanocrystal samples provided through collaboration with Vadim Cherezov (Scripps) for lipidic mesophase crystallizations, Petra Fromme (U. Arizona) for crystals amenable to XFEL analysis, and David Stokes (NYU) for 2D nanocrystals for structure determination by ED. Combined SONICC and synchrotron XRD measurements will be performed at the APS using a prototype capable of combined SHG imaging and focused "minibeam" XRD. Although the primary purposes of the present studies are to fundamentally advance membrane protein nanocrystal detection, close collaboration with Formulatrix will help lower the barriers to rapidly transition he findings of these inquiries to high-throughput commercial platforms for routine protein nanocrystal detection should the proposed efforts prove successful.