Investigating How Inter-domain Interactions Influence The Protein Folding Process

在20世纪60年代早期，安芬森奠定了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是由其氨基酸序列决定的。然而，这忽略了结构域间相互作用在多结构域蛋白折叠中的作用。根据定义，蛋白质结构域能够独立折叠成其功能折叠，但邻近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定量的分子水平描述：a)结构域间相互作用如何促进多结构域蛋白质的整体稳定性；B)这些结构域间相互作用如何影响蛋白质折叠机制。我建议通过研究酿酒酵母中Otu1酶作为一种范例多结构域酶的折叠来研究这些相互作用的意义。Otu1是一种含有348个氨基酸的蛋白质，它能催化与泛素有关的异肽键的切割。该蛋白形成三个不同的结构域：泛素样结构域，催化OTU结构域和假定的锌结合结构域。这三个结构域中的每一个都能够单独折叠成它们的生物活性构象。我的小组的研究计划，包括在任何给定时间的4个HQP，将包括以下步骤。******1)首先，通过监测各畴的热变性和化学变性分布，研究各畴的热力学性质。这些变性曲线可以用我实验室中存在的以下仪器来监测：差示扫描量热仪、圆二色光谱和荧光稳态。******2)研究各结构域的折叠动力学。蛋白质的折叠过程跨越了大量的时间尺度（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。在不同的蛋白质位置进行明智的氨基酸点突变，以便我们可以实时地局部探测蛋白质折叠；同时，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）使我们能够获得从纳秒到分钟的蛋白质折叠时间尺度。******3)动力学和热力学数据的结合将为我们提供每个区域折叠路径的完整图像。然后，我们将使用第1部分和第2部分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-结构域相互作用如何影响Otu1系统中的蛋白质稳定性和折叠过程。这些结果将为我们今后进一步研究其他多结构域酶的折叠提供一个蓝图。

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.