PROTEIN FOLDING DYNAMICS BY MASS SPECTROMETRY

阐明蛋白质折叠以获得其天然结构的机制仍然是生物化学中最具挑战性的问题之一。最近，许多疾病与可能的蛋白质错误折叠有关，随后是其功能的丧失/改变和/或聚集（淀粉样变性）。蛋白质的折叠/构象稳定性与其配体结合特性之间也有非常密切的关系。我们正在寻求进一步了解折叠过程的动力学，以及蛋白质和蛋白质配体复合物的构象稳定性。为此，我们将使用电喷雾（ESI）质谱（MS）来研究体外折叠过程。我们将利用溶液中的氢/氘（H/D）交换来探索不同条件下蛋白质的构象稳定性和折叠/展开动力学。这些实验将检测到瞬态填充的中间态，并以不同程度的骨干酰胺保护为特征。由于数据采集速率高，质谱法提供了一种简便的方法，可以在几十毫秒到几小时的时间尺度上解析这些中间体。关于蛋白质构象稳定性和折叠动力学的全局信息将由局部（残基特异性）信息补充。将采用几种方法来实现这一目标。在第一种方法中，蛋白质离子的碰撞激活解离（CAD）将用于测量局部氘含量作为交换时间的函数。应特别注意确保气相中的离子-分子过程（例如，氢搅合和/或与残余溶剂分子的H/D交换）不影响测量。这种方法对于在几十秒到几个小时的时间尺度上研究折叠事件特别有用，因为它允许进行实时监测。另一种方法是在一定时间内（通过降低溶液pH和温度）淬灭HID交换，并通过ESI/CAD或消化性消化分析H/D交换的程度，然后进行ESI MS分析。这将使我们的研究扩展到亚秒时间尺度。我们还将使用化学交联来阐明蛋白质中配体结合位点的拓扑结构，以及多结构域蛋白质中的结构域间相互作用。这些方法将用于研究几种蛋白质的折叠动力学和构象稳定性。配体与细胞维甲酸结合蛋白I （CRABP I）结合的分子机制将通过绘制载脂蛋白和全脂蛋白的能量格局来研究。我们还将研究转铁蛋白家族中的铁转运蛋白结合/释放铁离子的机制。所提出的研究将极大地促进我们对蛋白质折叠过程和转运蛋白在体内功能的机制的理解。

Elucidation of the mechanisms by which proteins fold to attain their native structure remains one of the most challenging problems in biochemistry. Recently, many diseases have been linked to possible protein misfolding followed by a loss/alteration of its function and/or aggregation (amyloidosis). There is also a very close relationship between the folding/conformational stability of proteins and their ligand- binding properties. We are seeking to further our understanding of the dynamics of the folding process, as well as conformational stability of proteins and protein-ligand complexes. To that end, we will use electrospray (ESI) mass spectrometry (MS) to study folding processes in vitro. We will employ hydrogen/deuterium (H/D) exchange in solution to probe the conformational stability and folding/unfolding dynamics of proteins under various conditions. Transiently populated intermediate states will be detected in these experiments and characterized by different degrees of backbone amide protection. Because of the high data acquisition rate, mass spectrometry offers a facile way to resolve these intermediates on a time scale from tens of msec to hours. The global information on the protein conformational stability and folding dynamics will be complemented by local (residue-specific) information. Several approaches will be used to achieve that goal. In the first approach, collisionally activated dissociation (CAD) of protein ions will be used to measure the deuterium content locally as a function of exchange time. Particular care will be taken to ensure that ion-molecular processes in the gas phase (e.g., hydrogen scrambling and/or H/D exchange with residual solvent molecules) do not influence the measurements. This approach will be particularly useful for studying the folding events on a time scale of tens of seconds to hours, as it allows real time monitoring to be carried out. In another approach, the HID exchange will be quenched at a certain time (by lowering the solution pH and temperature) and extent of the H/D exchange will be analyzed by using ESI/CAD or peptic digest followed by ESI MS analysis. This will allow us to extend our studies to a sub-second time scale. We will also use chemical cross-linking to elucidate the topology of ligand binding sites in proteins, as well as inter-domain interactions in multi-domain proteins. These methods will be applied to study folding dynamics and conformational stability of several proteins. Molecular mechanisms of ligand binding to Cellular Retinoic Acid Binding Protein I (CRABP I) will be studied by mapping the energy landscape for both apo- and holo-forms of the protein. We will also study mechanisms of ferric ion binding/release by iron transporting proteins from the transferrin family. The proposed studies will significantly further our understanding of the protein folding processes and the mechanisms by which transport proteins function in vivo.