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以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）产生足够量的用于分析的纯化蛋白，2）筛选多种可能的结晶条件，3）鉴定和分离用于X射线衍射分析的高质量晶体，和4）在X射线衍射测量之前精确定位尺寸小于~5 μ m的晶体。产生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以测定的条件的数量。此外，目前几乎没有可靠的现场方法可用于快速和非破坏性地评估蛋白质晶体质量和从衍射测量获得高分辨率结构的可能性，使得高分辨率结构的产生通常需要对候选晶体进行多轮试错分析。对于小晶体（<~5 μ m），简单地确定在同步辐射源衍射测量的晶体的位置是不平凡的。这些瓶颈可以潜在地通过所提出的开发手性晶体的二阶非线性光学成像（SONICC）来部分地解决，所述二阶非线性光学成像用于高选择性地检测初始晶体形成和晶体质量的初始评估。二次谐波产生是一种相干的非线性光学技术，在随机取向的组装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晶体，包括蛋白质的手性晶体，二次谐波产生是允许的。我们提出了SONICC检测和分析<5 μ m蛋白质晶体的仪器和方法的发展。如果成功的话，这些提出的技术有可能通过早期检测晶体形成，对预期衍射质量进行初始全光学评估，自动循环小于光学分辨率的晶体，以及在同步加速器源中进行衍射分析的高保真定位，实现对尺寸为1 μ m或更小的晶体的常规衍射分析。 实现这些目标需要一个调查小组的共同努力，每个小组都有互补的专门知识（图1）。将通过APS、Das和Simpson的SONICC团队之间的合作，对SONICC作为蛋白质晶体检测、表征和定位衍射分析的通用工具的验证进行评估。一旦普遍性得到确认，将通过布鲁克AXS、Jonathy Amy Facility for Chemical Instrumentation（JAFCI）和Simpson之间的合作，基于布鲁克CrystalHarvester平台构建利用SONICC从多角度非线性光学成像预测衍射质量的仪器。超高通量结晶筛选平台的开发也将由齐和辛普森同时进行，利用伯克纳米技术中心独特的微加工资源。 公共卫生关系： 手性晶体的二阶非线性光学成像（SONICC）将被探索作为一种通用的，灵敏的和高选择性的蛋白质晶体形成的检测方法。如果拟议项目成功实现特定目标，SONICC有可能直接解决大多数现代蛋白质结构测定工作常见步骤中的关键瓶颈，包括：1）快速测定不同的结晶条件，2）在提取到环中之前对晶体质量进行预筛选，3）使小于光学器件分辨率的晶体成环，以及4）将这种小晶体可靠地定位在紧密聚焦的同步加速器X射线源中用于衍射分析。来自普渡大学、阿贡国家实验室和布鲁克公司的跨学科、多机构研究人员团队将评估SONICC在常规蛋白质晶体检测、高通量和高通量结晶筛选读出以及集成到大型仪器平台中的普遍适用性。