PROTEIN FOLDING DYNAMICS BY MASS SPECTROMETRY

通过质谱分析蛋白质折叠动力学

• 批准号: 6387202
• 负责人: IGOR A KALTASHOV
• 金额: $ 17.49万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387202
• 关键词: mass spectrometry; molecular dynamics; protein folding; retinoid binding proteins; transferrin; mass spectrometry; molecular dynamics; protein folding; retinoid binding proteins; transferrin

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.

阐明蛋白质折叠以达到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，许多疾病已与可能的蛋白质错误折叠有关，然后是其功能和/或聚集（淀粉样变性）的损失/改变。蛋白质的折叠/构象稳定性与其配体结合特性之间也存在非常紧密的关系。我们正在寻求进一步了解折叠过程动力学，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）质谱法（MS）在体外研究折叠过程。 我们将在溶液中使用氢/氘（H/D）交换，以探测蛋白质在各种条件下的蛋白质的构象稳定性和折叠/展开动力学。在这些实验中将检测到瞬时填充的中间状态，并以不同程度的骨干酰胺保护为特征。 由于数据采集率很高，因此质谱法提供了一种从数十至小时的时间尺度上解决这些中间体的便利方法。 局部（残留特定）信息将补充有关蛋白质构象稳定性和折叠动力学的全局信息。 将使用几种方法来实现该目标。 在第一种方法中，蛋白质离子的碰撞激活解离（CAD）将用于在局部测量氘含量作为交换时间的函数。将要特别注意确保气相中的离子分子过程（例如，氢炒和/或h/d与残留溶剂分子交换）不会影响测量值。 这种方法对于在数十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视。 在另一种方法中，将在特定时间（通过降低溶液pH和温度）淬灭H/D交换的程度，然后使用ESI/CAD或Peptic Digest进行分析，然后进行ESI MS分析。 这将使我们能够将研究扩展到次秒时间范围。 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，以及多域蛋白质中的域间相互作用。这些方法将应用于研究几种蛋白质的折叠动力学和构象稳定性。 配体与细胞视黄酸结合蛋白I（CRABP I）结合的分子机制将通过映射蛋白质的apo-和Holo形式的能量景观来研究。 我们还将研究铁蛋白家族的铁转运蛋白的铁离子结合/释放的机制。拟议的研究将显着进一步了解我们对蛋白质折叠过程的理解以及转运蛋白在体内发挥作用的机制。