Ultrafast Nonlinear Optical Approaches toward High-Throughput Membrane Protein Na

超快非线性光学方法制备高通量膜蛋白 Na

• 批准号: 8824950
• 负责人: GARTH Jason SIMPSON
• 金额: $ 28.31万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8824950
• 关键词: Address; Arizona; Caliber; Characteristics; Collaborations; Complement; Crystallization; Dependence; Detection; Diffuse; Diffusion; Disease; Exhibits; Fluorescence; Fluorescence Polarization; Generations; Geometry; Health; Human; Image; Lasers; Measurement; Membrane Proteins; Methods; Noise; Optics; Pattern; Physiologic pulse; Polarization Microscopy; Process; Property; Proteins; Resolution; Roentgen Rays; Sampling; Scanning; Signal Transduction; Solutions; Source; Spectrum Analysis; Structure; Synchrotrons; System; Time; Validation; aqueous; base; cryogenics; crystallinity; electron diffraction; free-electron laser; imaging modality; improved; instrument; instrumentation; multi-photon; nano; nanocrystal; nanoscale; novel strategies; optical imaging; particle; protein structure; prototype; response; screening; second harmonic; tool; trend; two-photon

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.

描述（由申请人提供）：膜蛋白代表了结构测定中最具挑战性的蛋白质靶标之一，同时也是对人类健康最重要的蛋白质靶标之一。 X 射线源的重大新兴进步，包括 X 射线自由电子激光 (XFEL) 衍射、紧密聚焦同步加速器 XRD 的使用以及电子衍射 (ED) 仪器的改进，不可避免地减小了结构确定所需的晶体尺寸。蛋白质纳米晶体体系。这些趋势日益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。拟议工作的主要目标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，用于选择性筛选蛋白质纳米晶体的形成。将开发三种关键的新颖方法。首先，将开发和验证偏振依赖性 TPE-UVF，作为传统 SONICC 的补充，用于检测固定蛋白质晶体，例如在脂质中间相和低温条件下遇到的蛋白质晶体。有序结晶域产生与聚集体和溶剂化蛋白质不同的偏振依赖性 TPE-UVF 图案。第二，将开发 SHG 和 TPE-UVF 自相关方法，以提高扩散纳米晶体的检测限，针对水溶液生成的 2D 和 3D 纳米晶体。自相关依赖于对许多纳米晶体的组合弱响应的分析，以增强信号噪声，同时提供有关平移扩散的信息。最后，将使用偏振相关的 SHG 和 TPE-UVF 来改进非线性光学互相关光谱方法，用于选择性检测蛋白质纳米晶体旋转动力学。 TPE-UVF 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。所提出的方法的验证将结合使用聚焦 XRD、XFEL 衍射和 ED，并通过与用于脂质中间相结晶的 Vadim Cherezov（斯克里普斯）、Petra Fromme（美国亚利桑那州）合作提供的多样化且具有代表性的膜蛋白纳米晶体样品来进行。用于适合 XFEL 分析的晶体，David Stokes（纽约大学）用于通过 ED 确定二维纳米晶体的结构。 SONICC 和同步加速器 XRD 的组合测量将在 APS 上使用能够组合 SHG 成像和聚焦“迷你束”XRD 的原型进行。尽管本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障碍，将这些调查结果快速转化为用于常规蛋白质纳米晶体检测的高通量商业平台（如果拟议的努力证明）成功的。