Ultrafast Nonlinear Optical Approaches toward High-Throughput Membrane Protein Na

描述（由申请人提供）：膜蛋白是结构测定中最具挑战性的蛋白质靶标之一，也是对人类健康最重要的靶标。X射线源的主要新兴进展，包括使用X射线自由电子激光（XFEL）衍射，紧聚焦同步加速器XRD和电子衍射（艾德）仪器的改进，已无情地减少了蛋白质结构测定所需的晶体尺寸。这些趋势日益导致蛋白质衍射筛选的主要瓶颈，其重要性只会随着这些新的衍射能力的不断进步而增加。拟议努力的主要目标将是开发基于超快激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的形成。将开发三种关键的新方法。在第一，偏振依赖TPE-UVF将开发和验证作为常规SONICC的补充，用于检测不动的蛋白质晶体，如在介晶中间相和低温条件下遇到的那些。有序的结晶结构域产生不同于聚集体和溶剂化蛋白质的偏振依赖性TPE-UVF图案。第二，SHG和TPE-UVF自相关方法将被开发用于提高扩散纳米晶体的检测限，目标是从水溶液中产生的2D和3D纳米晶体。自相关依赖于对来自许多纳米晶体的组合弱响应的分析，用于信噪比增强，同时提供关于平移扩散的信息。最后，非线性光学互相关光谱方法将使用偏振相关的SHG和TPE-UVF选择性检测蛋白质的旋转动力学。TPE-UVF的偏振依赖性内的相关性将通过TPE-UVF和SONICC两者来保持自由扩散纳米晶体的晶体特异性检测。将使用聚焦XRD、XFEL衍射和艾德衍射的组合来验证所提出的方法，其中通过与Vadim Cherezov（Scripps）合作提供的具有代表性的不同膜蛋白质样品用于介晶中间相结晶，Petra Fromme（U. Arizona）用于XFEL分析的晶体，以及大卫斯托克斯（纽约大学）用于ED结构测定的2D纳米晶体。将在APS使用能够组合SHG成像和聚焦“微束”XRD的原型进行SONICC和同步加速器XRD组合测量。虽然目前研究的主要目的是从根本上推进膜蛋白的检测，但与Formulatrix的密切合作将有助于降低障碍，将这些调查的结果快速转移到高通量商业平台，用于常规蛋白的检测，如果拟议的努力证明是成功的。

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.