Post-Reductionist Protein Folding: Determining the In-cell Folding Energy Landsca

内质网(ER)是细胞的折叠工厂，每秒能够大量生产数千种分泌蛋白质。其中许多蛋白质的错误折叠会导致从囊性纤维化到肝硬变等一系列疾病。虽然可以使用多种生物物理技术在体外研究全长蛋白质，从而使生物物理学家能够详细描述他们的折叠能量格局，但在更具生理相关性的ER环境中研究蛋白质折叠面临着令人生畏的技术障碍。利用最近开发的、功能强大的单分子荧光技术，在含有荧光标记氨基酸的新生多肽链上，我们将确定内质网中新生多肽链延长时的构象演变，以及与内质网中的伴侣和修饰酶相互作用如何调节构象空间。其目标是获得关于不断增长的新生链条折叠情况的详细信息，与试管实验中提供的数据相媲美。内质网驻留蛋白和新生链间的共翻译和翻译后相互作用可能会平滑折叠能量格局，偏向于错误折叠和易于聚集的中间产物。这种折叠格局的重塑对于高接触顺序的蛋白质尤其重要，即链末端部分之间的许多接触。我们将关注一个重要的分泌性蛋白家族，抑制性丝氨酸，它折叠成具有高接触顺序的天然结构。有趣的是，蛇形原生状态在能量上处于亚稳定状态，准备改变为不同的结构。Serpins通过调节参与包括血液凝固和炎症在内的关键生理过程的蛋白水解酶，发挥着重要的生物学作用。许多与肝硬变和肺气肿等疾病相关的蛇针突变导致内质网中的蛇针聚集。通过研究内质网中的丝氨酸折叠并将其与体外折叠进行比较，我们将确定与内质网驻留蛋白的相互作用和疾病相关突变是如何改变折叠格局的。这项研究的结果将为蛇类如何错误折叠以及它们在体外和体内的能量景观如何不同提供前所未有的洞察。这项工作还将为科学家研究内质网中的蛋白质折叠提供一个容易获得的实验工具箱，以及一个治疗血清病和其他内质网折叠疾病的潜在治疗策略的平台。

The endoplasmic reticulum (ER) is a folding factory for the cell, able to churn out thousands of secretory proteins per second. Misfolding of a number of these proteins results in diseases ranging from cystic fibrosis to liver cirrhosis. While full-length proteins can be studied in vitro using a myriad of biophysical techniques, thus allowing biophysicists to characterize in detail their folding energy landscapes, studying protein folding in the more physiologically relevant ER environment presents daunting technical obstacles. Using recently developed, powerful single molecule fluorescence techniques on nascent polypeptide chains that contain fluorescently-labeled amino acids, we will determine the conformational evolution as a nascent polypeptide chain elongates in the ER and how conformational space is modulated by interactions with the chaperones and modifying enzymes resident in the ER. The goal is to obtain detailed information on the folding landscape of the growing nascent chain, rivaling the data available in test-tube experiments. Co- and post-translational interactions between the ER-resident proteins and nascent chains likely smooth the folding energy landscape, biasing against misfolded and aggregation-prone intermediates. This remodeling of the folding landscape is particularly important for proteins with high contact order, i.e. many contacts between distal parts of the chain. We will focus on an important family of secreted proteins, the inhibitory serpins, which fold into native structures with high contact order. Intriguingly serpin native states are energetically metastable, poised to change to a different structure. Serpins play crucial biological roles by regulating proteases involved in key physiological processes including blood coagulation and inflammation. A number of serpin mutations, associated with diseases such as liver cirrhosis and emphysema (together called 'serpinopathies'), lead to serpin aggregation in the ER. By studying serpin folding in the ER and comparing it to in vitro folding, we will determine how the folding landscape is altered by interactions with ER-resident proteins and by disease-associated mutations. The results of this research will provide unprecedented insight into how serpins misfold and how their energy landscapes differ in vitro and in vivo. This work will also provide a readily accessible experimental toolbox for scientists studying protein folding in the ER as well as a platform for potential therapeutic strategies to treat serpinopathies and other ER folding diseases.