Nonlinear Optical Imaging for Guiding Protein Structure Determination

用于指导蛋白质结构测定的非线性光学成像

• 批准号: 7768362
• 负责人: GARTH Jason SIMPSON
• 金额: $ 36.15万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7768362
• 关键词: Address; Binding; Biological Assay; Chemicals; Collaborations; Complex; Crystal Formation; Crystallization; Detection; Development; Devices; Dimensions; Disease; Early Diagnosis; Enzymes; Generations; Goals; Human; Imagery; Laboratories; Location; MJD1 protein; Measurement; Methods; Microfabrication; Microscope; Nanotechnology; Optics; Photons; Positioning Attribute; Printing; Process; Property; Protein Analysis; Proteins; Qi; Regulation; Research Personnel; Resolution; Resources; Roentgen Rays; Screening procedure; Site; Slide; Solutions; Source; Structure; Synchrotrons; System; Techniques; Twin Multiple Birth; UCHL1 gene; Universities; Validation; X ray diffraction analysis; X-Ray Diffraction; base; beamline; experience; instrument; instrumentation; mutant; optical imaging; protein structure; prototype; public health relevance; response; second harmonic; tool

DESCRIPTION (provided by applicant): Protein structure dictates function. Despite numerous advances in the development of high-throughput and ultrahigh-throughput platforms for protein crystallization screening for structure determination, major challenges remain including: 1) generating sufficient quantities of purified protein for analysis, 2) screening of a multitude of possible crystallization conditions, 3) identifying and isolating high-quality crystals for x-ray diffraction analysis, and 4) accurate positioning of crystals less than ~ 5 ¿m in dimension prior to X-ray diffraction measurements. The labor-intensive process of generating purified protein for crystallization screening often limits the number of conditions that can be assayed. Furthermore, few reliable on-site methods are currently available for rapidly and nondestructively assessing protein crystal quality and the likelihood of achieving high-resolution structures from diffraction measurements, such that the generation of high-resolution structures often requires multiple rounds of trial and error analysis on candidate crystals. For small crystals (<~5 ¿m), simply identifying the locations of the crystals for diffraction measurements at synchrotron sources is nontrivial. These bottlenecks can potentially be addressed in part through the proposed development of second order nonlinear optical imaging of chiral crystals (SONICC) for highly selective detection of incipient crystal formation and initial assessment of crystal quality. Second harmonic generation is a coherent nonlinear optical technique that disappears by symmetry in randomly oriented assemblies and in most achiral materials, but is bulk-allowed for the overwhelming majority of chiral crystals, including those of proteins. We proposed the development of instrumentation and methods for SONICC detection and analysis of <5 ¿m protein crystals. If successful, these proposed techniques have the potential to enable routine diffraction analysis of crystals ~1 ¿m in dimension or smaller, through early detection of crystal formation, initial all-optical assessment of anticipated diffraction quality, automated looping of crystal smaller than the optical resolution, and high-fidelity positioning in the synchrotron source for diffraction analysis. Realization of these goals will require the combined efforts of a team of investigators, each with complementary expertise (Figure 1). Validation of SONICC as a general tool for protein crystal detection, characterization, and positioning for diffraction analysis will be assessed through collaborative efforts between the SONICC Team at APS, Das, and Simpson. Once the generality is confirmed, instrumentation utilizing SONICC for predicting diffraction quality from multiple-angle nonlinear optical imaging will be constructed based on a Bruker CrystalHarvester platform through collaboration between Bruker AXS, the Jonathon Amy Facility for Chemical Instrumentation (JAFCI), and Simpson. Development of ultrahigh-throughput crystallization screening platforms will also be concurrently pursued by Qi and Simpson, taking advantage of unique microfabrication resources available in the Birck Nanotechnology Center. PUBLIC HEALTH RELEVANCE: Second-order nonlinear optical imaging of chiral crystals (SONICC) will be explored as a general, sensitive and highly selective detection approach for protein crystal formation. If the proposed project is successful in achieving the Specific Aims, SONICC has the potential to directly address key bottlenecks in steps common to most modern protein structure determination efforts, including: 1) rapidly assaying diverse crystallization conditions, 2) prescreening of crystal quality prior to extraction into a loop, 3) looping of crystals smaller than the resolution of the optics, and 4) reliably positioning such small crystals in tightly focused synchrotron X-ray sources for diffraction analysis. An interdisciplinary, multi-institutional team of investigators from Purdue University, Argonne National Laboratories, and Bruker will assess the general applicability of SONICC for routine protein crystal detection, for readout in high-throughput and ultrahigh throughput crystallization screenings, and for integration into larger instrument platforms.

描述（由申请人提供）： 蛋白质结构决定功能。尽管在用于蛋白质结晶筛选和确定结构的高通量和超高通量平台的开发方面取得了许多进展，但仍然存在主要挑战，包括：1) 产生足够数量的纯化蛋白质用于分析，2) 筛选多种蛋白质。可能的结晶条件，3) 识别和分离高质量晶体以进行 X 射线衍射分析，以及 4) 精确定位小于 ~ 5 ¿在 X 射线衍射测量之前，生成用于结晶筛选的纯化蛋白质的劳动密集型过程通常限制了可分析的条件的数量。此外，目前很少有可靠的现场方法可用于快速、非破坏性评估。蛋白质晶体质量以及通过衍射测量获得高分辨率结构的可能性，因此高分辨率结构的生成通常需要对候选晶体进行多轮试验和错误分析，对于小晶体（<~5 µm），只需简单地进行。确定用于同步加速器源衍射测量的晶体位置并非易事，可以通过拟议开发手性晶体二阶非线性光学成像（SONICC）来部分解决这些瓶颈，以高度选择性地检测初始晶体形成和初始评估。二次谐波产生是一种相干非线性光学技术，在随机取向的组件和大多数非手性材料中由于对称性而消失，但对于绝大多数手性材料是批量允许的。我们建议开发用于 SONICC 检测和分析 <5 ¿ 的仪器和方法。如果成功，这些提出的技术有可能实现晶体的常规衍射分析〜1 ¿ m 或更小的尺寸，通过晶体形成的早期检测、预期衍射质量的初始全光学评估、小于光学分辨率的晶体的自动循环以及用于衍射分析的同步加速器源中的高保真定位来实现这些目标。需要一组研究人员的共同努力，每个研究人员都具有互补的专业知识（图 1），SONICC 作为蛋白质晶体检测、表征和衍射分析定位的通用工具的验证将通过各个研究人员之间的合作进行评估。 APS、Das 和 Simpson 的 SONICC 团队一旦确认通用性，将通过 Bruker AXS、Jonathon Amy 设施之间的合作，基于 Bruker CrystalHarvester 平台构建利用 SONICC 预测多角度非线性光学成像衍射质量的仪器。 Qi 和 Simpson 还将利用独特的微加工资源，同时开发超高通量结晶筛选平台。伯克纳米技术中心提供。 公共卫生相关性： 将探索手性晶体二阶非线性光学成像（SONICC）作为蛋白质晶体形成的通用、高灵敏度和选择性检测方法如果拟议项目成功实现特定目标，SONICC 有潜力直接解决关键问题。大多数现代蛋白质结构测定工作中常见步骤的瓶颈，包括：1) 快速测定不同的结晶条件，2) 在提取到环中之前预筛选晶体质量，3) 小于晶体分辨率的晶体循环光学，4) 将这种小晶体可靠地定位在紧密聚焦的同步加速器 X 射线源中进行衍射分析，来自普渡大学、阿贡国家实验室和布鲁克的跨学科、多机构研究人员团队将评估 SONICC 的常规适用性。蛋白质晶体检测，用于高通量和超高通量结晶筛选中的读数，以及集成到更大的仪器平台中。