Nonlinear Optical Imaging for Guiding Protein Structure Determination

描述(由申请人提供)： 蛋白质结构决定功能。尽管用于结构确定的高通量和超高通量蛋白质结晶筛选平台的开发取得了许多进展，但主要的挑战包括：1)产生足够数量的用于分析的纯化蛋白质，2)筛选多种可能的结晶条件，3)识别和分离用于X-射线衍射分析的高质量晶体，以及4)在X-射线衍射测量之前精确定位尺寸小于5？m的晶体。生产用于结晶筛选的纯化蛋白的劳动密集型过程往往限制了可以检测的条件的数量。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，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错分析。对于较小的晶体(&lt；~5？m)，简单地确定晶体的位置以便在同步加速器源上进行衍射测量是不容易的。这些瓶颈可以通过手性晶体二阶非线性光学成像(SONICC)的拟议发展而被部分解决，该成像用于高选择性地检测初始晶体的形成和晶体质量的初步评估。二次谐波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，但对绝大多数手性晶体是整体允许的，包括蛋白质的晶体。我们提出了SONICC检测和分析蛋白质晶体的仪器和方法的发展。如果成功，这些建议的技术有可能通过及早发现晶体形成、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中的高保真定位，来实现对尺寸为1？m或更小的晶体的常规衍射分析。实现这些目标将需要一个调查小组的共同努力，每个小组都有互补的专业知识(图1)。将通过APS、DAS和Simpson的SONICC团队的合作努力，评估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有效性。一旦一般性得到确认，利用SONICC从多角度非线性光学成像预测衍射质量的仪器将通过Bruker AXS、Jonathon AMY化学仪器设施(JAFCI)和Simpson之间的合作，基于Bruker CrystalHarvester平台构建。齐和辛普森还将利用伯克纳米技术中心现有的独特微制造资源，同时开发超高通量结晶筛选平台。 公共卫生相关性： 手性晶体的二阶非线性光学成像(SONICC)将作为一种通用、灵敏和高选择性的蛋白质晶体形成检测方法。如果建议的项目成功地实现了特定目标，SONICC有可能通过大多数现代蛋白质结构测定工作中常见的步骤直接解决关键瓶颈，包括：1)快速分析不同的结晶条件，2)在提取到循环之前对晶体质量进行预筛选，3)将小于光学分辨率的晶体循环，以及4)将这种小晶体可靠地定位在紧密聚焦的同步辐射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、多机构的研究人员团队将评估SONICC在常规蛋白质晶体检测、高通量和超高通量结晶筛选中的读数以及集成到更大仪器平台中的一般适用性。

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.