Structural Biology of Cancer Related Membrane Proteins Expressed in P. Pastoris

描述（由申请人提供）：细胞分化、粘附和运动、增殖和凋亡是对细胞外环境的反应。膜蛋白检测环境刺激，并引发决定细胞命运的分子和遗传反应。当这些膜蛋白调控的过程发生故障时，癌症就产生了。尽管人膜蛋白在癌症生物学中起着核心作用，但目前还没有这些蛋白的高分辨率结构。我们的目标是解决在理解癌症分子机制方面的这一空白。我们将开展癌症相关膜蛋白（CRIMPs）结构生物学的全面研究。我们选择了涉及信号通路、细胞-细胞相互作用和运输的膜蛋白靶点，这些靶点是癌症生物学的组成部分。拟议研究的目标不是解决所有目标crips的结构。然而，我们所提出的方法和大而多样的目标集的结合使得求解一些卷曲结构变得现实。任何靶点的结构都将对癌症生物学的理解做出重大贡献。虽然很难预测膜蛋白结构在药物设计中的效用，但治疗潜力是我们选择靶标的重要因素，而CRIMPs的结构可能促进治疗药物或方案的开发。我们还将获得关于人膜蛋白在巴氏酵母中的异源表达的有价值的数据。在拟议的研究过程中，我们将完成迄今为止最全面的关于人膜蛋白在巴氏酵母中的表达的分析。本课题的具体研究目的是：1。鉴定适于结晶的与癌症相关的人膜蛋白。我们将根据癌症的相关性和预测疾病的程度选择60种结构和功能不同的膜蛋白。选择无序程度有限的CRIMPs将提高结晶成功的概率。2. 评价60种不同肿瘤相关膜蛋白在毕赤酵母中的异源表达。我们将使用新颖的Gateway¿1/2载体，10 ml帕斯德酵母生长，点印迹和小规模洗涤剂增溶实验来快速评估目标CRIMPs的异源表达和洗涤剂溶解度。所有通过增溶的实验将在每批12个目标上进行。3. 解决癌症相关膜蛋白的结构，过度表达和形成良好的晶体，而没有明显的修饰。我们将使用一种新的膜蛋白溶解度筛选来确定结晶目标的最佳浓度。我们将使用结晶机器人设置1024个条件，120微升蛋白质（每次实验~100 nl）。我们将使用实验室编写的软件和流体处理机器人来优化结晶。我们将使用自动板成像仪来观察和记录结晶实验。我们将解决形成高质量晶体的卷曲结构。

DESCRIPTION (provided by applicant): Cell differentiation, adhesion and motility, proliferation and apoptosis occur in response to the extracellular environment. Membrane proteins detect environmental stimuli, and elicit molecular and genetic responses that determine cell fate. Cancer results when these membrane-protein-orchestrated processes malfunction. Despite the central roles of human membrane proteins in cancer biology, no high-resolution structures of these proteins exist. Our goal is to address this gap in the understanding of the molecular mechanisms of cancer. We will initiate a comprehensive study of the structural biology of cancer-related membrane proteins (CRIMPs). We selected membrane protein targets involved in signaling pathways, cell-cell interactions, and transport that are integral to cancer biology. The goal of the proposed research is not to solve the structures of all of the target CRIMPs. However, the combination of our proposed methods and the large, diverse target set makes solving several CRIMP structures realistic. Structures of any of the targets will contribute significantly to the understanding of cancer biology. While it is difficult to predict the utility of membrane protein structures in drug design, therapeutic potential was an important factor in our target selection, and structures of CRIMPs may facilitate therapeutic drug or protocol development. We will also generate valuable data on the heterologous expression of human membrane proteins in P. pastoris. During the course of the proposed research we will complete the most comprehensive analysis to date on the expression of human membrane proteins in P. pastoris. The specific aims of the proposed research are: 1. Identify cancer related human membrane proteins that are amenable to crystallization. We will select 60 structurally and functionally diverse membrane proteins based on cancer relevance and degree of predicted disorder. Selecting CRIMPs with limited disorder will enhance the probability of successful crystallization. 2. Evaluate the heterologous expression of sixty diverse cancer related membrane proteins in Pichia pastoris. We will use novel Gateway¿1/2 vectors, 10 ml P. pastoris growths, dot-blots, and small scale detergent solubilization experiments to rapidly evaluate the heterologous expression and detergent solubility of target CRIMPs. All experiments through solubilization will be performed on batches of twelve targets. 3. Solve the structures of cancer-related membrane proteins that overexpress and form good crystals without significant modification. We will use a novel membrane protein solubility screen to determine the optimal concentrations of targets for crystallization. We will use a crystallization robot to set up 1024 conditions with 120 microliters of protein (~100 nl per experiment). We will use lab-written software and a fluid-handling robot to optimize crystallization. We will use an automated plate imager to view and document crystallization experiments. We will solve the structures of CRIMPs that form high quality crystals.