Project 4 (Goethe University of Frankfurt)

4.1.项目4(Détsch)。MPS和MP靶材的无细胞技术 4.3.背景和意义 确定MPS三维结构的主要障碍之一是产生足够数量的蛋白质。细菌细胞和真核细胞(酵母、昆虫细胞、哺乳动物细胞)都被用于表达MPS，但成功往往有限。特别是，真核MPS很难获得，这也反映在到目前为止只确定了大约10个真核MPS的结构。MPS在细胞系统中的表达受到几个问题的影响：合成蛋白的靶向和转位到正确的膜目的地，运输系统的过载和插入膜的毒性效应。 无细胞(CF)表达系统为MPS的规模化生产提供了一种全新的途径。这些系统不仅克服了基于细胞的表达系统的上述问题，而且还提供了相当多的额外好处。首先，翻译反应的开放性允许许多不同的化合物直接加入反应中，如蛋白酶和核糖核酸酶抑制剂、配体或伴侣。对于基于核磁共振的MPS的生产，无细胞表达系统提供了更多的优势。由于缺乏代谢紊乱氨基酸(AA) 在几乎任何组合中都可以进行特定类型的标记，这使得尽管具有有限的化学位移分散特性的S共振蛋白质的有效标记方案的开发成为可能(Klammt等人，2004年；Reckel等人，2008年；Trbovic等人，2005年)。因此，可以在不到两天的时间内产生用于核磁共振波谱结构分析的MPS样品。 一种独特而引人入胜的选择是在特定的疏水环境中生产可溶性MPS。在超过临界胶束浓度(CMC)的情况下，CF系统可以容忍几种洗涤剂，而蛋白质产量不会显著下降。加入此类洗涤剂可直接生成胶束增溶的MPS。这些MPS从未形成沉淀物或必须从含有刺激性洗涤剂的膜中提取。特别是，Brij系列的洗涤剂已被证明对生产大量胶束增溶的MPS非常有用。不幸的是，这些洗涤剂不能用于液态核磁共振研究，因为蛋白质-胶束复合体太大，导致非常宽和重叠的峰。然而，洗涤剂可以交换，例如通过将MP固定在柱子上并用含有所选洗涤剂的溶液洗涤。 在不同的洗涤剂和脂质体中使用MPS的经验表明，它们在胶束中的结构不如在脂双层中定义得好，这可能会对它们的稳定性和功能产生影响。脂质体将是MPS的理想天然环境，因为许多MPS，包括在无细胞系统中表达的MPS，已经被证明在这种环境中具有功能。不幸的是，脂质体太大，不适合液态核磁共振波谱，因此有必要寻找替代的天然环境。最近，双胞体和纳米盘被认为是疏水环境，它们足够小，可以用于液态核磁共振波谱，但同时更好地模拟了脂类双层的环境。在这 我们建议建立协议，使我们能够使用我们的CF表达系统将MPS直接表达到这样的疏水环境中。 获得大量标记的MPS并不是MP结构研究的唯一先决条件。这些蛋白质还必须具有功能。与大多数情况下只采用单一折叠状态的可溶性蛋白质不同，MPS可以有多种不同的状态，这取决于其疏水环境的确切组成。例如，与昆虫细胞表达的内皮素B受体(A GPCR)的结合分析表明，只有5-20%的纯化受体能够结合配体。测试(无细胞)表达的MPS在不同洗涤剂/脂类成分中的功能对于获得任何有意义的结构信息是必不可少的。因此，我们将建立结合和竞争分析以及基于功能G蛋白的分析来评估表达蛋白的状态。 最后，我们基于我们的CF生产方法开发了不同的标签方案，使我们能够以非常有效的方式获得MPS的主干分配。我们将确定不同MPS的核磁共振溶液结构，包括我们表达筛选的γ-分泌酶复合体的成分和GPCR靶标。

4.1. PROJECT 4 (D¿tsch). Cell-free Technology for MPs and MP targets 4.3. BACKGROUND AND SIGNIFICANCE One of the main obstacles to determining the 3-D structure of MPs is the production of sufficient quantities of proteins. Both bacterial and eukaryotic cells (yeast, insect cells, mammalian cells) have been used for the expression of MPs, but success if often limited. In particular eukaryotic MPs are difficult to obtain, which is also reflected by the fact that only ca. 10 structures of eukaryotic MPs have so far been determined. Expression of MPs in cellular systems suffers from several problems: targeting and translocation of the synthesized protein to the correct membrane destination, overloading of the transport system and toxic effects upon insertion into the membrane. Cell-free (CF) expression systems offer a completely new approach to the preparative scale production of MPs. These systems not only overcome the above mentioned problems of cell-based expression systems, they also provide a considerable number of additional benefits. First, the open nature of the translation reactions allows the addition of many different compounds, such as protease and RNAse inhibitors, ligands or chaperones directly into the reaction. For the production of MPs for NMR-based structure determination cell-free expression systems offer even more advantages. Due to the lack of metabolic scrambling amino acid (AA) type-specific labeling is possible in almost any combination, enabling the development of efficient labeling protocols for the assignment of the protein¿s resonances despite the limited chemical shift dispersion that is characteristic of α-helical MPs (Klammt et al., 2004; Reckel et al., 2008; Trbovic et al., 2005). Samples of MPs for the structural analysis by NMR spectroscopy can therefore be generated in less than two days. A unique and fascinating option is the CF production of soluble MPs into defined hydrophobic environments. Several detergents are tolerated by the CF system at concentrations above their critical micellar concentration (CMC) without significant decreases in protein yields. Addition of such detergents produces micelle-solubilized MPs directly. These MPs have never formed a precipitate or had to be extracted from a membrane with harsh detergents. In particular, detergents of the Brij family have proven to be very useful for the CF production of large amounts of micelle-solubilized MPs. Unfortunately these detergents cannot be used for liquid state NMR investigations since the protein-micelle complex is too large, leading to very broad and overlapping peaks. Detergents, however, can be exchanged, for example by immobilizing the MP on a column and washing it with a solution containing the detergent of choice. The experience with MPs in different detergents and liposomes has suggested that their structure in micelles is less well defined than in lipid bilayers which could have consequences for their stability and function. Liposomes would be the ideal ¿native-like¿ surrounding for MPs since many MPs ¿ including MPs expressed in cell-free systems - have proven to be functional in this environment. Unfortunately liposomes are too large for liquid state NMR spectroscopy, necessitating the search for alternative ¿native like environments¿. Recently bicelles and nanodiscs have been suggested as hydrophobic environments that are small enough for liquid state NMR spectroscopy but at the same time mimic better the environment of a lipid bilayer. In this project we propose to establish protocols that enable us to directly express MPs into such hydrophobic surroundings using our CF expression system. Obtaining large amounts of labeled MPs is not the only prerequisite for structural studies of a MP. These proteins also have to be functional. In contrast to soluble proteins that in most cases adopt only a single folded state, MPs can have many different states depending on the exact composition of their hydrophobic surrounding. Binding assays with insect-cell expressed endothelin B receptor (a GPCR) for example have shown that only 5-20% of purified receptor is capable of binding a ligand. Testing (cell free) expressed MPs for functionality in different detergent/lipid compositions is essential for obtaining any meaningful structural information. We will therefore establish binding and competition assays as well as functional G-protein based assays to assess the state of the expressed proteins. Finally, we have developed different labeling schemes based on our CF production approach that allow us to obtain the backbone assignment of MPs in a very efficient manner. We will determine the NMR solution structures of different MPs, including components of the y-secretase complex and GPCR targets of our expression screen.