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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Specialized Center--Cooperative Agreements (U54)
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Special Emphasis Panel (ZGM1-CBB-3)
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Harvard University
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