Investigating How Inter-domain Interactions Influence The Protein Folding Process

在20世纪60年代早期，Anfinsen奠定了已经成为蛋白质生物化学基石的教条的基础：即蛋白质的天然折叠由其氨基酸序列决定。然而，这忽略了结构域间相互作用在多结构域蛋白质折叠中的作用。根据定义，蛋白质结构域能够独立地折叠成其功能性折叠，但是相邻结构域的存在也必须影响蛋白质折叠。我的研究计划的主要目的是提供一个定量的分子水平的描述：a）域间的相互作用如何有助于多结构域蛋白质的整体稳定性; B）这些域间的相互作用如何影响蛋白质折叠机制。我建议通过研究酵母菌Otu 1的折叠来研究这些相互作用的意义。酿酒酵母作为范例多结构域酶。 Otu 1是一种含有348个氨基酸的蛋白质，催化泛素的异肽键断裂。该蛋白形成三个不同的结构域：泛素样结构域、催化OTU结构域和推定的Zn结合结构域。这三个结构域中的每一个都能够分别折叠成它们的生物活性构象。我的小组的研究计划，在任何给定的时间包括4个HQP，将包括以下步骤。 1)首先，通过监测每个结构域的热变性和化学变性概况来研究每个结构域的热力学性质。这些变性配置文件可以监测与以下仪器存在于我的实验室：差示扫描量热法，圆二色光谱和荧光稳态。 2)研究每个结构域的折叠动力学。蛋白质的折叠过程跨越大量的时间尺度（微秒到分钟），因此我们将使用分子生物学和独特的光谱技术相结合来研究结构域折叠和展开的动力学。明智的氨基酸点突变将在不同的蛋白质位置，使我们可以在本地探测蛋白质折叠在真实的时间;而我最近获得的最先进的动力学仪器（停止流动，温度跳跃光谱和时间分辨荧光）使我们能够访问蛋白质折叠的时间跨度从纳秒到分钟。 3)结合动力学和热力学数据将为我们提供一个完整的图片折叠途径的每个域。然后，我们将使用第1部分和第2部分中开发的协议来研究完整蛋白质的折叠动力学和热力学。 因此，我们将量化结构域之间的相互作用如何影响蛋白质的稳定性和折叠过程中的Otu 1系统。这些结果将为我们提供一个蓝图，以扩大我们的调查，研究其他多域酶的折叠在未来。

In the early 1960s, Anfinsen laid down the foundations of the dogma that has become the cornerstone of protein biochemistry: namely, that the native fold of a protein is determined by its amino acid sequence. However, this neglects the role that inter-domain interactions play in the folding of multi-domain proteins. Protein domains by definition are capable of folding into their functional folds independently, but the presence of neighboring domains must also affect the protein fold. The major aim of my research program is to provide a quantitative molecular-level description of: a) how inter-domain interactions contribute to the overall stability of multi-domain proteins; b) how these inter-domain interactions influence the protein folding mechanism. I propose to investigate the significance of these interactions by studying the folding of the enzyme Otu1 from the yeast S. cerevisiae as a paradigm multidomain enzyme. Otu1 is a protein containing 348 amino acids and catalyzes the cleaving of isopeptide bonds involving ubiquitins. This protein forms three distinct domains: the ubiquitin-like domain, the catalytic OTU domain, and the putative Zn-binding domain. Each of these three domains are capable of folding separately into their biologically active conformations. The research program for my group, that includes 4 HQP at any given time, will include the following steps. 1) First, study the thermodynamic properties of each domain by monitoring the thermal and chemical denaturation profile of each domain. These denaturation profiles can be monitored with the following instrumentation that exists in my laboratory: differential scanning calorimetery, circular dichroism spectroscopy and fluorescence steady-state. 2) Study the folding kinetics of each domain. Folding processes in proteins span a large number of timescales (microseconds to minutes), therefore we will study the kinetics of domain folding and unfolding using a combination of molecular biology and unique spectroscopy techniques. Judicious amino acid point mutations will be made in different protein locations so that we can locally probe protein folding in real time; while, my recently acquired state-of-the-art kinetic instrumentation (stopped flow, temperature jump spectroscopy and time-resolved fluorescence) allows us to access protein folding timescales spanning nanoseconds to minutes. 3) The combination of kinetic and thermodynamic data will provide us with a complete picture of the folding pathway of each domain. We will then use the protocols developed in parts 1 and 2 to study the folding kinetics and thermodynamics of the complete protein. We shall thus quantify how domain-domain interactions influence protein stability and the folding process in the Otu1 system. These results will provide us with a blue print to extend our investigations to study the folding of other multi-domain enzymes in the future.