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: 8888526
• 负责人: GARTH Jason SIMPSON
• 金额: $ 26.61万
• 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 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8888526
• 关键词: Adopted; Affect; Birefringence; Caliber; Chemicals; Communities; Crystal Formation; Crystallization; Crystallography; Custom; Data Collection; Defect; Detection; Development; Dimensions; Disease; Dose; Dyes; Equilibrium; Failure; Feedback; Fluorescence; Fluorescence Microscopy; Foundations; Generations; Goals; Growth; Image; In Situ; Kinetics; Laboratories; Lasers; Legal patent; Length; Licensing; Measurement; Methods; Microscopy; Mothers; Optics; Peer Review; Photons; Population; Positioning Attribute; Preparation; Procedures; Production; Property; Protein Analysis; Proteins; Protocols documentation; Registries; Resolution; Resources; Roentgen Rays; Sampling; Signal Transduction; Source; Staging; Structure; Synchrotrons; Technology; Time; Twin Multiple Birth; Uncertainty; United States National Institutes of Health; X ray diffraction analysis; X-Ray Diffraction; base; beamline; contrast imaging; distilled alcoholic beverage; fluorescence microscope; free-electron laser; high reward; high risk; image guided; imaging platform; improved; instrument; instrumentation; intercalation; journal article; nano; nanocrystal; optical imaging; protein structure; public health relevance; screening; second harmonic; structural biology; success; synchrotron radiation; theories; two-photon; ultraviolet

 DESCRIPTION (provided by applicant): High-resolution protein structures determined by X-ray diffraction require the production of high-quality, well-ordered crystals. The generation of such crystals typically involves the identification of initial crystallization conditions, followedby optimization to produce well-ordered crystals, and subsequent X-ray diffraction, most commonly performed at synchrotron facilities. Each step in this pipeline can typically require weeks or months to assess success or failure in iterative optimizations, limited by a combination of slow kinetics for protein nucleation and growth in order to generate large well-ordered single crystals, and limited access to synchrotron facilities for assessing crystal quality and performing data collection. We propose the use of nonlinear optical imaging to rapidly inform the final three key experimental steps of initial crystal formation, optimization of crystal quality prior to diffractin, and synchrotron X-ray diffraction analysis. For the first goal, intercalation of protein crystals wth "SHG-phores" is proposed for enhancing the range and sizes of crystals detectable by SHG, with proof-of-concept measurements suggesting enhancement factors of 1000-fold are achievable. In situ assessment of crystal quality is proposed for rapid optimization based the use of polarization-dependent SHG imaging for identification of multi-domain, twinned, and highly mosaic crystals. Finally, instrumentation to enable rapid serial crystallography of micro- and nano-crystalline showers are proposed based on integration of synchrotron XRD with multi-modal confocal reflectance, brightfield transmittance, birefringence, two-photon excited ultraviolet fluorescence (TPE-UVF), and UV-SHG, all acquired simultaneously at up to video rate and with perfect image registry. Using this combined measurement suite, we aim to enable routine and confident detection of crystals 1-5mm in diameter to enable diffraction measurements with a 1 mm collimated source. The proposed efforts build on a foundation laid during the previous initial cycle of NIH support. SHG and TPE-UVF microscopy first proposed in our previous period of support are now established and widely used methods within the crystallography community. Previous support directly contributed to 35 peer-reviewed journal articles, 66 presentations, and 3 issued patents. Our technology has been licensed, and a commercial SHG/TPE-UVF microscope is now available for protein crystal screening. In addition, a custom first-generation nonlinear optical imaging instrument has been integrated into a macromolecular crystallography beamline at Argonne National Laboratory.

 描述(申请人提供)：由X射线衍射确定的高分辨率蛋白质结构需要生产高质量、有序的晶体。这类晶体的产生通常包括确定初始结晶条件，然后优化以产生有序的晶体，以及随后的X射线衍射，最常见的是在同步加速器设施中进行。该流水线中的每一步通常需要数周或数月来评估迭代优化中的成功或失败，这受到蛋白质成核和生长的缓慢动力学的组合的限制，以便产生大的有序的单晶， 以及用于评估晶体质量和进行数据收集的同步加速器设施的使用受限。我们建议使用非线性光学成像来快速地告知最后三个关键实验步骤：初始晶体形成、晶体质量优化和同步辐射X射线衍射分析。对于第一个目标，提出了插入具有“倍频荧光”的蛋白质晶体，以扩大倍频可探测到的晶体的范围和大小，概念验证测量表明可以实现1000倍的增强倍数。基于偏振相关倍频成像用于识别多域、孪晶和高度镶嵌的晶体，提出了基于偏振相关倍频成像的晶体质量的现场评估以快速优化。最后，基于同步加速器X射线衍射仪与多模共焦反射率、明场透过率、双折射、双光子激发紫外荧光(TPE-UVF)和UV-SHG的集成，提出了能够实现微米和纳米晶体簇射快速连续结晶的仪器，所有这些都以高视频速率和完美的图像配准同时获得。使用这一组合测量套件，我们的目标是能够对直径1-5 mm的晶体进行常规和可靠的检测，从而能够在1 mm准直源的情况下进行衍射测量。拟议的努力建立在前一个国家卫生研究院最初支持周期中奠定的基础上。倍频显微镜和TPE-UVF显微镜最初是在我们之前的支持时期提出的，现在已经建立起来，并在结晶学社区内广泛使用。以前的支持直接促成了35篇同行评议的期刊文章、66篇演示文稿和3项专利颁发。我们的技术已经获得许可，商业SHG/TPE-UVF显微镜现在可以用于蛋白质晶体筛选。此外，一台定制的第一代非线性光学成像仪器已经集成到阿贡国家实验室的大分子晶体光束线中。