Applications of Cell-Free Synthesized Membrane Protein Precipitates.

Cell-free protein expression systems are new core platforms for membrane protein synthesis. Expression in the presence of supplied artificial hydrophobic environments such as nanomembranes or micelles allows the co-translational solubilization and folding of membrane proteins. In the absence of hydrophobic compounds, the synthesized membrane proteins quantitatively precipitate, while frequently still retaining a significant part of folded structural elements. This so-called precipitate-forming cell-free (P-CF) expression mode is a very effective and reliable approach for numerous applications. Even from complex membrane proteins such as G-protein coupled receptors or large transporters, significant amounts of such precipitates can be synthesized within few hours. The precipitates can be solubilized in detergents or reconstituted into membranes for subsequent structural or functional analysis. Harsh denaturation and refolding procedures as known from the treatment of bacterial inclusion bodies are usually not required.This strategy is particularly interesting for applications requiring large amounts of membrane protein or fast access to a sample. It is further an excellent tool for the production of membrane protein antigens suitable for antibody generation. The purification of the precipitates in downstream processing is streamlined as only few proteins from the cell-free lysate may co-precipitate with the synthesized membrane protein. For most applications, a one-step affinity chromatography by taking advantage of small purification tags attached to the membrane protein target is sufficient. We give an overview on current applications of P-CF precipitates and describe the underlying techniques in detail. We furthermore provide protocols for the successful crystallization and NMR analysis of P-CF synthesized membrane proteins exemplified with the diacylglycerol kinase (DAGK). In addition, we describe the functional characterization of a P-CF synthesized large eukaryotic transporter.

Modified lipid and protein dynamics in nanodiscs.

For membrane protein studies, nanodiscs have been shown to hold great potential in terms of preparing soluble samples while maintaining a lipid environment. Here, we describe the differences in lipid order and protein dynamics in MSP1 nanodiscs compared to lamellar preparations by solid-state NMR. For DMPC, an increase of the dipolar C-H lipid acyl chain order parameters in nanodiscs is observed in both gel- and liquid crystalline phases. Incorporating proteorhodopsin in these nanodiscs resulted in a significantly longer rotating frame spin-lattice relaxation time for (13)C leerzeichen and better cross polarisation efficiency due to restricted protein dynamics. A comparison of (13)C-(13)C correlation spectra revealed no structural differences. The incorporation of proteorhodopsin into nanodiscs has been optimised with respect to detergent and to protein/scaffold protein/lipid stoichiometries. Its functional state was probed by time-resolved optical spectroscopy revealing only minor differences between lamellar and nanodisc preparations. Our observations show remarkable dynamic effects between membrane proteins, lipids and scaffold protein. The potential use of nanodiscs for solid-state NMR applications is discussed.

A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein TBsmr.

TBsmr is a secondary active multidrug transporter from Mycobacterium tuberculosis that transports a plethora of compounds including antibiotics and fluorescent dyes. It belongs to the small multidrug resistance (SMR) superfamily and is structurally and functionally related to E. coli EmrE. Of particular importance is the link between protein function, oligomeric state and lipid composition. By freeze fracture EM, we found three different size distributions in three different lipid environments for TBsmr indicating different oligomeric states. The link of these states with protein activity has been probed by fluorescence spectroscopy revealing significant differences. The drug binding site has been probed further by (19)F-MAS NMR through chemical labeling of native cysteine residues showing a water accessible environment in agreement with the alternating access model.

How to investigate interactions between membrane proteins and ligands by solid-state NMR.

Solid-state NMR is an established method for biophysical studies of membrane proteins within the lipid bilayers and an emerging technique for structural biology in general. In particular magic angle sample spinning has been found to be very useful for the investigation of large membrane proteins and their interaction with small molecules within the lipid bilayer. Using a number of examples, we illustrate and discuss in this chapter, which information can be gained and which experimental parameters need to be considered when planning such experiments. We focus especially on the interaction of diffusive ligands with membrane proteins.

Morphological differences between ß(2) -microglobulin in fibrils and inclusion bodies.

Over expression of proteins in E. coli frequently results in the production of inclusion bodies. Although ß(2) -microglobulin frequently forms fibrillar structures, our studies reveal significant differences between the protein in fibrils and inclusion bodies. This suggests that the formation of fibrils in inclusion bodies is dependent on the propensity of the protein to form fibrillar structures.

Membrane domain structures of three classes of histidine kinase receptors by cell-free expression and rapid NMR analysis.

NMR structural studies of membrane proteins (MP) are hampered by complications in MP expression, technical difficulties associated with the slow process of NMR spectral peak assignment, and limited distance information obtainable for transmembrane (TM) helices. To overcome the inherent challenges in the determination of MP structures, we have developed a rapid and cost-efficient strategy that combines cell-free (CF) protein synthesis, optimized combinatorial dual-isotope labeling for nearly instant resonance assignment, and fast acquisition of long-distance information using paramagnetic probes. Here we report three backbone structures for the TM domains of the three classes of Escherichia coli histidine kinase receptors (HKRs). The ArcB and QseC TM domains are both two-helical motifs, whereas the KdpD TM domain comprises a four-helical bundle with shorter second and third helices. The interhelical distances (up to 12 A) reveal weak interactions within the TM domains of all three receptors. Determined consecutively within 8 months, these structures offer insight into the abundant and underrepresented in the Protein Data Bank class of 2-4 TM crossers and demonstrate the efficiency of our CF combinatorial dual-labeling strategy, which can be applied to solve MP structures in high numbers and at a high speed. Our results greatly expand the current knowledge of HKR structure, opening the doors to studies on their widespread and pharmaceutically important bacterial signaling mechanism.