High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics.

Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics.

Synthetic Biology-Based Solution NMR Studies on Membrane Proteins in Lipid Environments.

Although membrane proteins are in the focus of biochemical research for many decades the general knowledge of this important class is far behind soluble proteins. Despite several recent technical developments, the most challenging feature still is the generation of high-quality samples in environments suitable for the selected application. Reconstitution of membrane proteins into lipid bilayers will generate the most native-like environment and is therefore commonly desired.

Protein labeling strategies for liquid-state NMR spectroscopy using cell-free synthesis.

Preparation of a protein sample for liquid-state nuclear magnetic resonance (NMR) spectroscopy analysis requires optimization of many parameters. This review describes labeling strategies for obtaining assignments of protein resonances. Particular emphasis is placed on the advantages of cell-free protein production, which enables exclusive labeling of the protein of interest, thereby simplifying downstream processing steps and increasing the availability of different labeling strategies for a target protein.

Insights into Cotranslational Membrane Protein Insertion by Combined LILBID-Mass Spectrometry and NMR Spectroscopy.

Cotranslational insertion of membrane proteins into defined nanoparticle membranes has been developed as an efficient process to produce highly soluble samples in native-like environments and to study lipid-dependent effects on protein structure and function. Numerous examples of the structural and functional characterization of transporters, ion channels, or G-protein-coupled receptors in cotranslationally formed nanodisc complexes demonstrate the versatility of this approach, although the basic underlying mechanisms of membrane insertion are mainly unknown. We have revealed the first aspects of the insertion of proteins into nanodiscs by combining cell-free expression, noncovalent mass spectrometry, and NMR spectroscopy.

Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology.

Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies.

From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins.

Nanodiscs and isotropic bicelles are promising membrane mimetics in the field of solution nuclear magnetic resonance (NMR) spectroscopy of integral membrane proteins (IMPs). Despite varied challenges to solution NMR studies of IMPs, we attribute the paucity of solution NMR structures in these environments to the inability of diverse IMPs to withstand detergent treatment during standard nanodisc and bicelle preparations. Here, we present a strategy that creates small isotropic bicelles from IMPs co-translationally embedded in large nanodiscs using cell-free expression.

Labeling of membrane proteins by cell-free expression.

The particular advantage of the cell-free reaction is that it allows a plethora of supplementation during protein expression and offers complete control over the available amino acid pool in view of concentration and composition. In combination with the fast and reliable production efficiencies of cell-free systems, the labeling and subsequent structural evaluation of very challenging targets, such as membrane proteins, comes into focus. We describe current methods for the isotopic labeling of cell-free synthesized membrane proteins and we review techniques available to the practitioner pursuing structural studies by nuclear magnetic resonance spectroscopy.

Time-shared experiments for efficient assignment of triple-selectively labeled proteins.

Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially.

Characterization of two distinct modes of drug binding to human intestinal fatty acid binding protein.

The aqueous cytoplasm of cells poses a potentially significant barrier for many lipophilic drugs to reach their sites of action. Fatty acid binding proteins (FABPs) bind to poorly water-soluble fatty acids (FAs) and lipophilic compounds and facilitate their intracellular transport. Several structures of FA in complex with FABPs have been described, but data describing the binding sites of other lipophilic ligands including drugs are limited.

Examination of the role of intestinal fatty acid-binding protein in drug absorption using a parallel artificial membrane permeability assay.

Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally diverse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein - intestinal fatty acid-binding protein (I-FABP).