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，从而大大加快蛋白质结构的测定与使用创新的肽酰胺氢交换技术提供的信息。在这次重新提交的文件中，我们提出了自审查以来进行的广泛的初步研究，这些研究直接解决了对我们先前提交的文件的所有保留意见。 需要以越来越高的通量（HT）速度确定高分辨率结构，以从根本上理解癌症相关蛋白质的修饰如何促进肿瘤发生和转移。不幸的是，HT晶体学的努力有一个单一的，主要的障碍：他们为少数目标蛋白质产生合适的晶体。失败蛋白质的松散的非结构化区域在这个问题中起着重要作用。 蛋白质内许多肽酰胺氢的交换速率由蛋白质在单个氨基酸尺度上的稳定性决定。我们已经开发了一种增强型的酰胺氢/氘交换质谱（DXMS），可以快速，精确地测量这种速率。我们建议DXMS数据可用于识别和定位蛋白质内的非结构化区域，从而指导修饰蛋白质的设计，其中选择性地去除这些区域。此外，许多蛋白质需要由结合伴侣提供的三级-四级接触，以诱导这些区域中的结构。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伴侣，从而实现集中的蛋白质结合伴侣共结晶努力。重要的是，对修饰蛋白质的重复DXMS研究可以快速确定它们保留原始蛋白质结构元素的程度。 在我们的R21年，我们将证明DXMS可以指导蛋白质结构的重新设计，足以使目标蛋白质的总体结晶成功率提高50%，并以高通量的速度实现这一目标。这一结果将确立DXMS加速目前HT晶体学工作的通量的能力，并且可能类似地增强常规特异性蛋白质聚焦晶体学的构建体定义，对于癌症相关蛋白质的结构研究具有明显的益处。 在我们的R33年，我们将建立一个晶体学专用的DXMS设施，并通过分析我们在结构基因组学联合中心的合作者研究的蛋白质靶点，进一步完善我们指导构建设计的能力，重点是那些与癌症相关的蛋白质。然后，这种构建-改进资源将作为一种社区服务广泛扩展到NCI资助的研究人员，以应用于常规和HT晶体学工作。

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.