Investigation of protein dynamics by mass spectrometry

描述(申请人提供)：建议的研究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。对具有重要功能的瞬时非天然蛋白质状态的详细和高度特异性的表征将依赖于结合溶液中氢/氢交换(HDX)、电喷雾离子化(ESI MS)和气相蛋白质离子碎裂的实验策略的开发。新的策略将通过结合使用HDX和(I)代表单一蛋白质状态的几个离子物种的同时碎裂和(Ii)天然和非天然蛋白质状态的共价捕获来提供构象特定的结构和动态信息。还将开发一种新的方法，通过选择性地监测极性侧链上的HDX来探测蛋白质核心的稳定性。正离子和负离子模式下的多阶段蛋白质离子碎裂与溶液相HDX相结合将被评估为描述具有多个二硫键的大(&gt；30 kDa)蛋白质的局部动力学的一种手段。这些新的策略将被用来加深我们对细胞维甲酸结合蛋白I和II(CRABP I和II)作为控制维甲酸(RA)结合和释放的关键因素的一过性紊乱的理解。将完成发生配体结合的非天然蛋白质状态的结构表征，随后将研究CRABP II与维甲酸受体(RAR)结合所诱导的动态事件，该RAR促进配体从运输蛋白向受体的转移。新的实验策略也将被应用于通过检测血清转铁蛋白(Tf)的动力学和金属结合特性以及转铁蛋白受体(TfR)对它们的调节来加深我们对通过转铁蛋白循环向细胞输送金属的理解。了解RAR/CRABP和TF/TFR相互作用的分子机制对于利用维甲酸的多效性和TF向肿瘤细胞运送细胞毒配体的能力的多种治疗策略的加强具有重要的价值。

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.