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，但对人类健康也是最重要的。x射线源的主要新进展，包括使用x射线自由电子激光（XFEL）衍射，紧密聚焦同步加速器XRD，以及电子衍射（ED）仪器的改进，不可避免地减少了蛋白质纳米晶体结构测定所需的晶体尺寸。这些趋势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不断进步，其重要性只会增加。本研究的主要目标是开发基于超快激光源非线性光相互作用的测量工具，用于选择性筛选蛋白质纳米晶体的形成。将开发三个关键的新方法。首先，极化依赖的TPE-UVF将被开发和验证，作为传统SONICC检测固定蛋白晶体的补充，例如在脂质中间期和低温条件下遇到的蛋白质晶体。有序的晶体结构域产生极化依赖的TPE-UVF模式，不同于聚集体和溶剂化蛋白质。其次，将开发SHG和TPE-UVF自相关方法，以提高扩散纳米晶体的检出限，针对水溶液中产生的二维和三维纳米晶体。自相关依赖于分析来自许多纳米晶体的联合弱响应来增强信号对噪声，同时提供有关平移扩散的信息。最后，将利用偏振依赖的SHG和TPE-UVF的非线性光学互相关光谱方法来选择性检测蛋白质纳米晶体的旋转动力学。TPE-UVF的偏振依赖性内的相关性将保留TPE-UVF和SONICC对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ED相结合的方法对所提出的方法进行验证，并与Vadim Cherezov （Scripps）合作提供各种具有代表性的膜蛋白纳米晶体样品进行脂质中间相结晶，Petra Fromme （U. Arizona）合作提供适合XFEL分析的晶体。联合SONICC和同步加速器XRD测量将在APS上进行，使用能够结合SHG成像和聚焦“微束”XRD的原型机。虽然目前研究的主要目的是从根本上推进膜蛋白纳米晶体检测，但与Formulatrix的密切合作将有助于降低将这些调查结果快速转化为常规蛋白质纳米晶体检测的高通量商业平台的障碍，如果提议的努力被证明是成功的。

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.