EAGER: Coherent Guest Protein Organization Inside Host Protein Crystals

EAGER：宿主蛋白晶体内一致的客体蛋白组织

• 批准号: 1645015
• 负责人: Christopher Snow
• 金额: $ 10万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1645015&HistoricalAwards=false
• 关键词: EAGER; Coherent; Guest; Protein; Organization

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Snow at Colorado State University, is developing a new approach for observing proteins in atomic detail. Structure determination of proteins is a key bottleneck for understanding and engineering protein molecules. The project aims to circumvent the challenges of growing conventional protein crystals, by taking the unprecedented step of attaching target proteins to specific sites within pre-existing, crystalline scaffolds. Once guest proteins are attached to the scaffold crystal, they need to adopt a structure to be visible via X-ray diffraction. This is a highly risky step but the pay-off will be huge too. Success will open the door for an accessible and economical alternative to conventional structure determination methods for protein studies.The current dominant method for structure determination in atomic detail is X-ray crystallography, but this method requires a brute force search through non-physiological solution conditions, looking for the ?needle-in-a-haystack? condition in which the target protein crystallizes. Unfortunately, despite exhaustive screening, most proteins of interest (~70%) do not form crystals. Other proteins are difficult to obtain in sufficient quantities to make the attempt. Finally, the crystals that do grow reveal a structure adopted under artificial conditions, a single snapshot that dramatically under-represents protein mobility. The motivational insight for this project is the recognition that materials diffract X-rays if they consist of a highly-ordered, repeating lattice, but that the lattice need not be composed only of target protein. Supported by this EAGER grant, Professor Snow is tryong to demonstrate that guest proteins anchored to the crystalline scaffold can adopt a coherent structure visible via X-ray diffraction and then correlate the guest structure observed as a function of solution conditions. To achieve this goal, the Snow group first prepare thiol bearing scaffold crystals and covalently install guest proteins using disulfide bonds or homobifunctional crosslinkers. The resulting crystals are screened for coherent guest organization via solvent substitution and x-ray diffraction. When necessary, the scaffolds are further engineered via chemical modification or genetic mutation. The project provides an educational training opportunity in cutting-edge areas of bionanotechnology, chemical biology, applied biophysics, and molecular modeling.

在化学系化学测量和成像项目的支持下，科罗拉多州立大学的斯诺教授正在开发一种观察蛋白质原子细节的新方法。蛋白质结构的确定是理解和改造蛋白质分子的关键瓶颈。该项目旨在通过采取前所未有的步骤，将目标蛋白质附着到预先存在的晶体支架内的特定位点，从而规避传统蛋白质晶体生长的挑战。一旦客体蛋白附着在支架晶体上，它们需要采用一种通过X射线衍射可见的结构。这是一个高风险的步骤，但回报也将是巨大的。成功将打开一个方便和经济的替代传统的结构测定方法的蛋白质studies.The当前占主导地位的方法结构测定原子细节是X射线晶体学，但这种方法需要通过非生理溶液条件下的蛮力搜索，寻找？大海捞针目标蛋白质结晶的条件。不幸的是，尽管进行了详尽的筛选，但大多数感兴趣的蛋白质（约70%）不形成晶体。其他蛋白质很难获得足够的数量来进行尝试。最后，生长的晶体揭示了在人工条件下采用的结构，这是一个明显低估蛋白质流动性的单一快照。该项目的动机是认识到，如果材料由高度有序的重复晶格组成，则材料会吸收X射线，但晶格不一定只由靶蛋白组成。在EAGER基金的支持下，Snow教授试图证明锚定在晶体支架上的客体蛋白可以通过X射线衍射观察到一种连贯的结构，然后将观察到的客体结构与溶液条件相关联。为了实现这一目标，Snow团队首先制备了含有巯基的支架晶体，并使用二硫键或同双功能交联剂共价安装客体蛋白。通过溶剂取代和X射线衍射筛选所得晶体的相干客体组织。必要时，支架通过化学修饰或基因突变进一步工程化。该项目提供了生物纳米技术，化学生物学，应用生物物理学和分子建模等前沿领域的教育培训机会。