Enhanced Crystallography of Cancer-Implicated Proteins

描述(由申请人提供)： 我们的目标是提高设计蛋白质晶体结构的能力，从而利用创新的肽酰胺氢交换技术提供的信息大幅加快蛋白质结构的确定。在这次重新提交中，我们介绍了自审查以来进行的广泛的初步研究，这些研究直接涉及对我们先前提交的材料的所有保留意见。 为了从根本上了解癌症相关蛋白的修饰如何促进肿瘤的发生和转移，需要以越来越高的吞吐量(HT)速度确定高分辨率结构。不幸的是，羟色胺的结晶学努力有一个主要的障碍：他们为一小部分目标蛋白质产生合适的晶体。失败蛋白质的软性、非结构化区域在这个问题上起着主要作用。 蛋白质中许多肽胺氢的交换率是由蛋白质在单个氨基酸范围内的稳定性决定的。我们已经开发了一种增强型酰胺氢/氚交换质谱仪(DXMS)，可以快速而准确地测量这种速率。我们认为，DXMS数据可以用来识别和定位蛋白质中的这些非结构区域，从而指导选择性地去除这些区域的修饰蛋白质的设计。此外，许多蛋白质需要结合伙伴提供的三-四级接触，才能在这些区域诱导结构。对于这些蛋白质，DXMS可用于快速选择提供所需稳定接触的结合伙伴，从而允许有重点的蛋白质结合伙伴共结晶努力。重要的是，重复对修饰蛋白质(S)进行DXMS研究可以快速确定它们保留了原始蛋白质的结构元素的程度。 在我们的R21年，我们将证明DXMS可以充分指导蛋白质结构的重新设计，从而使目标蛋白质的总结晶成功率提高50%，并以高通量的速度做到这一点。这一结果将建立DXMS的能力，以加快目前的HT结晶学工作的速度，并可能类似地增强传统的、特定蛋白质聚焦的结晶学的结构定义，对癌症相关蛋白质的结构研究具有明显的好处。 在我们的R33年中，我们将建立一家专门用于结晶学的DXMS设施，并通过分析我们在结构基因组联合中心的合作者研究的蛋白质靶标，进一步完善我们指导结构设计的能力，重点是那些与癌症相关的蛋白质靶标。然后，这种构造精炼资源将作为社区服务广泛扩展到NCI资助的研究人员，以应用于传统和HT结晶学工作。

DESCRIPTION (provided by applicant): We aim to enhance the ability to design protein crystallographic constructs and, thereby, substantially speed protein structure determination with the use of information provided by innovative peptide amide hydrogen exchange techniques. In this resubmission, we present extensive preliminary studies, performed since review, that directly address all reservations regarding our prior submission. Determination of high-resolution structure at an increasingly high throughput (HT) pace is required for a fundamental understanding of how modifications of cancer- implicated proteins can promote oncogenesis and metastasis. Unfortunately, HT crystallographic efforts have a single, dominating roadblock: they produce suitable crystals for a small minority of target proteins. Floppy, unstructured regions of failed proteins play a major role in this problem. The exchange rates of the many peptide amide hydrogens within a protein are determined by the protein's stability at the individual amino acid scale. We have developed an enhanced form of amide hydrogen/deuterium exchange-mass spectrometry (DXMS) that can rapidly and precisely measure such rates. We propose that DXMS data can be used to identify and localize such unstructured regions within a protein and thereby guide the design of modified protein in which such regions are selectively removed. Furthermore, many proteins require tertiary-quaternary contacts, provided by binding partners, to induce structure in such regions. For these proteins, DXMS can be used to rapidly select binding partners that provide the needed stabilizing contacts, allowing focused protein-binding partner co-crystallization efforts. Importantly, repeat DXMS study of the modified protein(s) can rapidly determine how well they have retained the structured elements of the original protein. In our R21 year, we will demonstrate that DXMS can guide the re-design of protein constructs sufficiently to produce a 50% increase in overall crystallization success rates for target proteins, and do this at a high throughput pace. This result will establish the ability of DXMS to speed throughput of present HT crystallographic efforts, and likely similarly enhance construct definition for conventional, specific-protein focused crystallography, with obvious benefits for the structural study of cancer related proteins. In our R33 years we will establish a crystallography-dedicated DXMS facility and further refine our ability to guide construct design by analysis of the protein targets studied by our collaborators at the Joint Center for Structural Genomics, with an emphasis on those with cancer-relevance. This construct-refinement resource will then be broadly extended as a community service to NCI-funded investigators for application to both conventional and HT crystallographic efforts.