Investigation of protein dynamics by mass spectrometry

描述（由申请人提供）：拟议研究的重点是结构可塑性和大尺度动力学作为蛋白质功能的决定因素。详细和高度特异性表征功能重要的瞬态非天然蛋白质状态将依赖于结合溶液中氢/氘交换（HDX），电喷雾分离（ESI MS）和气相蛋白质离子碎片化的实验策略的发展。新策略将通过使用HDX结合(i)代表单一蛋白质状态的几个离子物种的同时断裂和（ii）天然和非天然蛋白质状态的共价捕获来提供特定的构象结构和动态信息。此外，还将开发一种通过选择性地监测HDX极性侧链来探测蛋白质核心稳定性的新方法。在正离子和负离子模式下，结合溶液HDX，多阶段蛋白质离子碎裂将被评估为表征具有多个二硫键的大（bbb30 kDa）蛋白质内部局部动力学的一种手段。这些新策略将有助于我们进一步了解细胞维甲酸结合蛋白I和II （CRABP I和II）的短暂性紊乱是控制维甲酸（RA）结合和释放的关键因素。配体结合发生的非天然蛋白状态的结构表征将完成，随后将研究CRABP II与视黄酸受体（RAR）结合诱导的动态事件，该动态事件促进配体从转运蛋白转移到受体。新的实验策略还将通过检测血清转铁蛋白（Tf）的动力学和金属结合特性及其受转铁蛋白受体（TfR）的调节，进一步应用于我们对通过转铁蛋白周期向细胞传递金属的理解。了解RAR/CRABP和Tf/TfR相互作用的分子机制，对于提高利用视黄酸的多效性和Tf向恶性细胞递送细胞毒性配体的能力的各种治疗策略将是非常宝贵的。

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.