Membrane protein reconstitution

Fernandez C, Wuthrich K (2003) NMR solution structure determination of membrane proteins reconstituted in detergent micelles. FEBS Lett 555 144-150... 

Most attractive are solid-state NMR studies of membrane proteins, in their native membranes, or membrane proteins reconstituted into synthetic lipid vesicles with defined lipid composition. Several integral membrane proteins are present in sufficiently high concentrations in the native membranes (> 25% of total membrane protein) to offer samples concentrated enough to permit the application of solid-state NMR without... 

J-L Rigaud, D Levy, G Mosser, O Lambert. Detergent removal by non-polar polystyrene beads. Applications to membrane protein reconstitution and two-dimensional crystallization. Eur Biophys J 27 305-319, 1998. 

Many of the metabolite uptake studies cited above rely on combined uptake and incorporation into starch. In order to separate uptake from incorporation, Schott et al.226 extracted amyloplast membrane proteins from potato tubers and reconstituted them into liposomes. These reconstituted liposomes transported Pi, triose phosphates and G6P in a counter-exchange mode. The liposomes were ineffective in the transfer of G1P uptake of ADP-Glc was not tested. Mohlmann et al.236 have used a proteoliposomic system to reconstitute plastid envelope proteins. In this system, ADP-Glc is transported in exchange for AMP. Thus the more widely studied plastid ATP/ADP transporter was not responsible for ADP-Glc uptake. More recently, Bowsher et al.237 reported that wheat endosperm amyloplasts membrane proteins reconstituted into proteoliposomes took up ADP-Glc in exchange for AMP and ADP. In addition, they showed that under conditions of ADP-Glc dependent starch biosynthesis, the efflux of ADP from intact amyloplasts was equal to that of ADP-Glc utilization by starch synthesis. The amyloplast membrane ADP-Glc/ADP transporter was a 38 000 molecular weight integral membrane protein.237... 

The highest resolution cryo-EM data has thus far been obtained for proteins present in 2-D crystals (a technique known as electron crystallography). This technique measures the structural features of membrane proteins reconstituted into 2-D crystals in the presence of lipid bilayers. In contrast to the 3-D crystals used in XRC, in which proteins are solubilized in detergent micelles that can disrupt crystal formation and reduce crystal quality, formation of 2-D crystals forces the membrane proteins to pack within a lipid bilayer, thus restoring their native environment. Another advantage of 2-D crystallization is that it requires very small amounts of protein (as opposed to NMR, which requires milligram quantities). 

Figure 3 Methods for supported bilayer formation and membrane protein reconstitution, (a) and (b) LB/LS method. A lipid monolayer is spread at the air-water interface of a Langmuir trough and transferred to a solid substrate while keeping the surface pressure constant. A second monolayer is transferred by horizontal apposition of the first transferred monolayer and collection of a counter-piece with spacers, (c) Direct VF method. Membrane vesicles are flown into a chamber with a clean surface substrate on top. After about an hour of incubation, they form a supported bilayer on the substrate and excess vesicles are flushed out. (d) LB/VF method. The procedures depicted in panels (a) and (c) are combined leading to an asymmetric bilayer with an asymmetric protein distribution. Although this method can also be performed without a polymer, it is shown here with the polymer transferred during the LB step.

Other proteins that have activities that correlate with the mesomorphic tendencies of the lipid bilayer include the vertebrate photoreceptor protein rhodopsin (42) and a dolichylphosphomannose synthase (43). The paucity of other examples reflects the lack of systematic studies. Membrane protein reconstitutions are generally difficult to perform, especially if the lipid composition is to be varied, and, therefore, are unlikely to be undertaken without good reason. Studies of correlations with lipid mesomorphic tendencies, stimulated by research such as that reported here, are now under consideration by several biochemical groups. Certainly, much more work is needed in this area. 

R 391 C. Fernandez and K. Wuethrich, NMR Solution Structure Determination of Membrane Proteins Reconstituted in Detergent Micelles , FEBS Lett., 2003,555,144... 

Ultrafast MAS experiments sensitivity can be enhanced through the use of low power sequences combined with paramagnetically enhanced relaxation times to reduce recycle delays, as well as proton detected experiments. In this work the sensitivity of and detected experiments applied to large membrane proteins reconstituted in lipids and packed in small MAS NMR rotors. This is an attractive prospect for samples of limited quantity, as this allows for a reduction in the amount of protein that needs to be produced without the necessity for increased experimental time. 

The low sensitivity inherent to both the static and MAS techniques of SS NMR spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Researchers demonstrated the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. They sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. ... 

Obtaining high-resolution NMR spectra of membrane proteins reconstituted in micelles. 

---