Investigation of protein dynamics by mass spectrometry

描述（由申请人提供）：拟议研究的重点是作为蛋白质功能决定因素的结构可塑性和大规模动力学。功能上重要的瞬时非天然蛋白质状态的详细和高度特异性表征将依赖于结合溶液中的氢/氘交换（HDX）、电喷雾离子化（ESI MS）和气相蛋白质离子片段化的实验策略的发展。新的策略将通过使用HDX结合（i）代表单一蛋白质状态的几种离子物质的同时片段化和（ii）天然和非天然蛋白质状态的共价捕获来提供符合者特异性结构和动态信息。还将开发一种通过在极性侧链上选择性地监测HDX来探测蛋白质核心稳定性的新方法。将评价正离子和负离子模式下的多阶段蛋白质离子片段化与溶液相HDX的组合，作为表征具有多个二硫键的大蛋白质（> 30 kDa）内局部动力学的方法。这些新的策略将被用来进一步了解细胞视黄酸结合蛋白I和II（CRABP I和II）作为一个关键因素控制视黄酸（RA）的结合和释放的短暂性障碍。将完成配体结合发生的非天然蛋白质状态的结构表征，然后研究CRABP II内由其与视黄酸受体（RAR）结合诱导的动态事件，这有助于配体从转运蛋白转移到受体。新的实验策略也将被应用到进一步了解金属传递到细胞通过转铁蛋白循环检查动力学和金属结合特性的血清转铁蛋白（Tf）和转铁蛋白受体（TfR）的调制。了解RAR/CRABP和Tf/TfR相互作用的分子机制，将是非常宝贵的增强各种治疗策略，利用视黄酸的多效性和Tf的能力，提供细胞毒性配体的恶性细胞。

DESCRIPTION (provided by applicant): The focus of the proposed research is on structural plasticity and large-scale dynamics as determinants of protein function. Detailed and highly specific characterization of functionally important transient non-native protein states will rely on development of experimental strategies combining hydrogen/deuterium exchange in solution (HDX), electrospray ipnization (ESI MS) and gas phase protein ion fragmentation. The new strategies will provide conformer-specific structural and dynamic information by using HDX in combination with (i) simultaneous fragmentation of several ionic species representing a single protein state and (ii) covalent trapping of native and non-native protein states. A new method to probe stability of protein cores by monitoring HDX selectively at polar side chains will also be developed. Multi-stage protein ion fragmentation in both positive and negative ion modes in combination with solution-phase HDX will be evaluated as a means of characterizing local dynamics within large (> 30 kDa) proteins with multiple disulfide bonds. These new strategies will be used to further our understanding of transient disorder in cellular retinoic acid binding proteins I and II (CRABP I and II) as a critical factor controlling binding and release of retinoic acid (RA). Structural characterization of non-native protein states through which ligand binding occurs will be completed, followed by investigation of the dynamic events within CRABP II induced by its binding to a retinoic acid receptor (RAR), which facilitate ligand transfer from the transport protein to the receptor. The new experimental strategies will also be applied to further our understanding of metal delivery to cells via the transferrin cycle by examining dynamics and metal-binding properties of serum transferrin (Tf) and their modulation by transferrin receptor (TfR). Understanding the molecular mechanisms of RAR/CRABP and Tf/TfR interactions will be invaluable for the enhancing of a variety of therapeutic strategies exploiting the pleiotropic properties of retinoic acid and the ability of Tf to deliver cytotoxic ligands to malignant cells.