A ß-barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL.

The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.

Probing Membrane Protein Insertion into Lipid Bilayers by Solid-State NMR.

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s-1 was determined for residues protected from exchange. Transfer within the protein, as tracked in the 15 N-1 H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s-1 for several resolved residues, explaining the site specificity.

Membrane functionalization of polymersomes: alleviating mass transport limitations by integrating multiple selective membrane transporters for the diffusion of chemically diverse molecules.

Recently, the interest in polymersomes as nanoreactors for synthetic applications has increased due to interesting proof-of-concept studies, indicating a versatile use of polymeric vesicles to compartmentalize complex reaction cascades. However, the low permeability of polymeric membranes and the requirement for a controlled mass transport across the compartment boundaries have posed a major limitation to the broad applicability of polymersomes for synthetic reactions. Current advances in the functional integration of membrane proteins (MPs) into poly(2-dimethylsiloxane)-based membranes have allowed the selective increase of the permeability for a controlled mass transport of the desired compounds across the membrane. Herein we demonstrate that polymer membranes are capable of harboring different MPs to alleviate the mass transport limitations of chemically diverse molecules, thereby enabling complex cascade reactions to be performed within the nanoreactors. The ability to functionalize the polymer membrane with multiple, highly selective MPs allows a reduction in mass transport limitations without abandoning compartmentalization of the reaction space on a low molecular mass level. As the model reaction, a two enzyme system consisting of a ketoreductase (KR) and a formate dehydrogenase was studied. For the transport of the hydrophobic substrate and product of the KR, the MPs AlkL, OmpW, OprG and TodX were investigated. For the transport of formate, OmpF, PhoE and FocA were used. AlkL showed the highest integration efficiency (39%) and a maximum of 120 AlkL molecules were successfully inserted into each polymersome. The highest channel-specific effects on the mass transfer were achieved using TodX and PhoE, respectively. The combination of both proteins led to an improvement of the space-time yield of the product (S)-pentafluorophenyl ethanol by 2.32-fold compared to nanoreactors without MPs.

Preparative refolding of small monomeric outer membrane proteins.

The outer membrane of gram-negative bacteria constitutes an important hurdle for the transport of hydrophobic molecules into the cell. Mass flux is often facilitated by various outer membrane proteins. These proteins are of biotechnological importance because they could help to improve the performance of whole-cell biocatalysts or be incorporated into artificial cell-like systems. The characterization and understanding of their transport properties greatly benefits from the possibility to express and purify these proteins. We investigated folding parameters for the refolding of four small monomeric outer membrane proteins from Escherichia coli (OmpW) and different pseudomonads (AlkL, OprG and TodX). To this aim we screened a number of inexpensive detergents and detergent concentrations, folding additives as well as protein concentrations. Interestingly, detergents with a C12 chain were most effective in promoting the folding reaction, particularly the negatively charged N-Lauroylsarcosine for OmpW, OprG and TodX as well as the zwitterionic N,N-Dimethyl-n-dodecylamine N-oxide (LDAO) for AlkL. The addition of 1 M urea (AlkL, OmpW), 0.1 M glutamate (OprG) or 0.1 M glycine (TodX) could further improve the folding efficiency. In order to be able to reproducibly produce larger amounts of the proteins, we then established the folding in a miniaturized stirred-tank reactor system combined with a liquid handler. This approach led to a near-complete refolding of OprG (96%), a very good folding of AlkL (84%) and OmpW (71%), only TodX folding was more variable with a final folding efficiency of 52%, all obtained at a final protein concentration of 0.5 g/L.

Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR.

Most commonly small outer membrane proteins, possessing between 8 and 12 ß-strands, are not involved in transport but fulfill diverse functions such as cell adhesion or binding of ligands. An intriguing exception are the 8-stranded ß-barrel proteins of the OmpW family, which are implicated in the transport of small molecules. A representative example is AlkL from Pseudomonas putida GPoI, which functions as a passive importer of hydrophobic molecules. This role is of high interest with respect to both fundamental biological understanding and industrial applications in biocatalysis, since this protein is frequently utilized in biotransformation of alkanes. While the transport function of AlkL is generally accepted, a controversy in the transport mechanism still exists. In order to address this, we are pursuing a structural study of recombinantly produced AlkL reconstituted in lipid bilayers using solid-state NMR spectroscopy. In this manuscript we present 1H, 13C and 15N chemical shift assignments obtained via a suite of 3D experiments employing high magnetic fields (1 GHz and 800 MHz) and the latest magic-angle spinning (MAS) approaches at fast (60-111) kHz rates. We additionally analyze the secondary structure prediction in comparison with those of published structures of homologous proteins.

Selective 1H-1H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR.

Very fast magic-angle spinning (MAS > 80 kHz) NMR combined with high-field magnets has enabled the acquisition of proton-detected spectra in fully protonated solid samples with sufficient resolution and sensitivity. One of the primary challenges in structure determination of protein is observing long-range 1H-1H contacts. Here we use band-selective spin-lock pulses to obtain selective 1H-1H contacts (e.g., HN-HN) on the order of 5-6 Å in fully protonated proteins at 111 kHz MAS. This approach is a major advancement in structural characterization of proteins given that magnetization can be selectively transferred between protons that are 5-6 Å apart despite the presence of other protons at shorter distance. The observed contacts are similar to those previously observed only in perdeuterated proteins with selective protonation. Simulations and experiments show the proposed method has performance that is superior to that of the currently used methods. The method is demonstrated on GB1 and a ß-barrel membrane protein, AlkL.