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。尽管在用于结构测定的蛋白质结晶筛选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 µm 的晶体。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、无损地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错分析。对于小晶体（<~5 µm），简单地识别晶体位置以在同步加速器源进行衍射测量是很重要的。这些瓶颈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，该成像用于高选择性检测初始晶体形成和晶体质量的初步评估。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晶体（包括蛋白质晶体）来说是批量允许的。我们提议开发用于 SONICC 检测和分析 <5 µm 蛋白质晶体的仪器和方法。如果成功的话，这些提出的技术有可能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，实现尺寸为~1μm或更小的晶体的常规衍射分析。 实现这些目标需要研究人员团队的共同努力，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通用工具的验证将通过 APS、Das 和 Simpson 的 SONICC 团队之间的合作进行评估。一旦普遍性得到确认，通过布鲁克 AXS、乔纳森艾米化学仪器设施 (JAFCI) 和辛普森之间的合作，将基于布鲁克 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 Birck 纳米技术中心独特的微加工资源，同时开发超高通量结晶筛选平台。

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.