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）：膜蛋白是结构确定最具挑战性的蛋白质靶标之一，但对人类健康最重要。 X射线源的主要新兴进步，包括使用X射线游离电子激光器（XFEL）衍射，紧密聚焦的同步加速器XRD以及电子衍射仪（ED）仪器的改善，可以不可避免地降低了将结构确定到蛋白质Nananocrystal Incemime中所需的晶体大小。这些趋势越来越多地导致蛋白质纳米晶筛查的主要瓶颈，其重要性只会随着这些新的衍射能力继续提高而增加。拟议工作的主要目标是基于从超快激光源的非线性光学相互作用开发测量工具，以选择性筛选蛋白质纳米晶形成。将开发三种关键的新颖方法。首先，将开发和验证依赖极化的TPE-UVF，作为对常规SonicC检测固定蛋白晶体的补充，例如在脂质中间酶和低温条件下遇到的蛋白质晶体。有序的晶体结构域产生依赖于极化的TPE-UVF模式，不同于聚集体和溶剂化蛋白。在第二个中，将开发SHG和TPE-UVF自相关方法，以改善扩散纳米晶体的检测极限，靶向由水溶液产生的2D和3D纳米晶体。自相关依赖于对许多纳米晶体的组合反应的分析，以提高信号，同时提供有关转化扩散的信息。最后，使用依赖极化的SHG和TPE-UVF来选择性检测蛋白质纳米晶体旋转动力学的选择性检测，将采用非线性光学互相关方法。 TPE-UVF的极化依赖性之间的相关性将通过TPE-UVF和SonicC保留晶体特异性检测，以自由扩散纳米晶体。将使用聚焦XRD，XFEL衍射和ED的组合进行验证，并与通过与Vadim Cherezov（Scripps）合作提供多种多样的和代表性的膜蛋白蛋白纳米晶体样品，以进行脂质中的中间酶结晶（U. Arizona and arok andy andy andy and arok am am an andy andy andy andy and arok am am am an andy andy andy andy andy） 2D纳米晶体用于ED结构测定。合并的SonicC和Synchrotron XRD测量将使用能够组合SHG成像和聚焦为“ MiniBeam” XRD的原型在AP上进行。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迅速过渡的障碍，但他发现这些询问的高通量商业平台可用于常规蛋白质纳米晶体检测的高通量商业平台。