DXMS-Facilitated Membrane Protein Construct Design/Cancer

描述（由申请人提供）： 许多与癌症有关的蛋白质是整合膜蛋白（IMP）。迫切需要改进用于生产IMP构建体的方法，以用于高分辨率结构测定工作。三年前，我们完成了十五年的努力，以开发肽酰胺氢/氘交换质谱法（DXMS）的性能的方法。在与结构基因组学联合中心（JCSG）的合作中，我们最近证明了DXMS可以精确地提供指导结晶良好的可溶性蛋白质结构设计所需的信息。NCI IMAT计划现在正在资助我们优化DXMS指导的可溶性癌症相关蛋白质的构建体设计（R33 CA099835）。 直到最近，我们认为成功的膜蛋白DXMS分析是不可能的，而且这项资助没有提到膜蛋白（IMP），也不支持它们的工作。 然而，本申请中描述的见解和初步研究现在使得有可能通过密集的开发工作，我们可以设计高度修改的方法，其将允许IMP的简易DXMS分析。膜蛋白DXMS的开发将极大地影响与癌症有关的IMP的结构生物学，这些IMP特别难以以可结晶形式制备。 最初的第1年开发工作将集中在整合素allbbs上，我对此有相当丰富的经验。整联蛋白广泛涉及癌细胞和癌症脉管系统生物学，并且原型allbbS整联蛋白的发现已被证明适用于理解所有整联蛋白。将在第2年通过研究Raymond Stevens博士（P.I.）提供的其他癌症相关IMP和子构建体，进一步完善和验证所得IMPDXMS方法。JCSG创新膜蛋白技术中心（JCIMPT） 一旦IMP-DXMS得到充分开发和验证，它将通过将这些方法与我们现在由NCI IMAT计划支持的可溶性蛋白DXMS资源整合，提供给研究癌症相关IMP的研究人员。因此，NCI在目前资助的DXMS工作中的投资将被IMP-DXMS开发所需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.