EAGER: Coherent Guest Protein Organization Inside Host Protein Crystals

在化学系化学测量与成像项目的支持下，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工程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，将目标蛋白质附着在预先存在的晶体支架的特定位置上，从而规避生长传统蛋白质晶体的挑战。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。成功将打开一扇大门，为传统的蛋白质结构测定方法的可访问和经济替代。目前确定原子细节结构的主要方法是x射线晶体学，但这种方法需要在非生理溶液条件下进行蛮力搜索，寻找“大海捞针”。目标蛋白结晶的状态。不幸的是，尽管进行了详尽的筛选，大多数感兴趣的蛋白质（约70%）都不能形成晶体。其他蛋白质很难获得足够的量来进行尝试。最后，确实生长的晶体揭示了在人工条件下采用的结构，这是一个显著低于蛋白质流动性的单一快照。这个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，就会衍射x射线，但晶格不一定只由目标蛋白质组成。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采用通过x射线衍射可见的连贯结构，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硫醇的支架晶体，并使用二硫键或同源双功能交联剂以共价方式安装客体蛋白。所得晶体通过溶剂取代和x射线衍射来筛选相干客体组织。必要时，通过化学修饰或基因突变进一步改造支架。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。

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.