DXMS-Facilitated Membrane Protein Construct Design/Cancer

描述(由申请人提供)： 许多与癌症相关的蛋白质是完整的膜蛋白(IMP)。迫切需要改进用于高分辨率结构确定工作中使用的IMP构建体的生产方法。三年前，我们完成了长达十五年的努力，以开发多肽酰胺氢/氢交换质谱仪(DXMS)的性能方法。在与联合结构基因组学中心(JCSG)的合作下，我们最近证明了DXMS可以准确地提供所需的信息，以指导设计结晶良好的可溶性蛋白质。NCI IMAT计划现在正在资助我们优化DXMS引导的可溶性癌症相关蛋白质(R33 CA099835)的结构设计。直到最近，我们还认为不太可能对膜蛋白进行成功的DXMS分析，而且这笔资助没有提到膜蛋白，也不支持对膜蛋白的研究。然而，本申请中描述的洞察力和初步研究现在使我们有可能通过密集的开发工作，设计出高度改进的方法，从而允许对IMP进行简便的DXMS分析。膜蛋白DXMS的开发将极大地影响与癌症相关的IMPS的结构生物学，这些IMPS特别难制备成结晶形式。最初的第一年开发工作将集中在整合素allbbS上，我在这方面有相当多的经验。整合素广泛存在于癌细胞和肿瘤血管生物学中，其原型allbbS整合素的发现已被证明适用于对所有整合素的理解。由此产生的IMPDXMS方法将在第二年通过对由NIH新资助的JCSG创新膜蛋白技术中心(JCIMPT)的Raymond Stevens，P.I.博士提供的其他与癌症相关的IMP和子体结构的研究来进一步改进和验证。一旦IMP-DXMS被完全开发和验证，它将通过将这些方法与我们目前由NCI IMAT计划支持的可溶性蛋白质DXMS资源整合起来，供研究癌症相关IMP的研究人员使用。因此，NCI对目前获得资助的DXMS工作的投资将被对IMP-DXMS发展所要求的相对适度的支持所极大地利用。

DESCRIPTION (provided by applicant): Many cancer-implicated proteins are integral membrane proteins (IMPs). There is a pressing need for improved methods for the production of IMP constructs for use in high-resolution structure determination efforts. Three years ago, we completed a fifteen- year effort to develop methods for the performance of peptide amide hydrogen/deuterium exchange- mass spectrometry (DXMS). In collaboration with the Joint Center for Structural Genomics (JCSG), we recently demonstrated that DXMS can provide precisely the information needed to guide the design of well-crystallizing constructs of otherwise poorly-crystallizing soluble proteins. The NCI IMAT program is now funding our efforts to optimize DXMS-guided construct design for soluble cancer-implicated proteins (R33 CA099835). Until recently, we thought it unlikely that successful DXMS analysis of membrane proteins would be possible, and this funded grant contains no reference to membrane proteins (IMPs), nor does it support work on them. However, insights and preliminary studies described in the present application now make it likely that, with intensive development work, we can devise highly modified methods that will allow the facile DXMS analysis of IMPs. Development of membrane protein DXMS will greatly impact the structural biology of cancerimplicated IMPs, which are particularly difficult to prepare in crystallizable form. Initial year 1 development efforts will focus on the integrin allbbS, with which I have had considerable experience. Integrins are widely implicated in cancer cell and cancer vasculature biology, and findings with the prototypic allbbS integrin have proven applicable to the understanding of all integrins. The resulting IMPDXMS methods will be further refined and validated in year 2 through study of additional cancer- relevant IMPs and daughter constructs provided by Dr. Raymond Stevens, P.I. of the newly NIH-funded JCSG Center for Innovative Membrane Protein Technologies (JCIMPT). Once IMP- DXMS has been fully developed and validated, it will be made available to investigators studying cancer-implicated IMPs, by integrating the methods with our soluble-protein DXMS resource now supported by the NCI IMAT program. Thus the NCI's investment in presently funded DXMS work will be greatly leveraged by the relatively modest support requested for the development of IMP-DXMS.