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. 

Doupnik CA, Davidson N, Lester HA et al (1997) RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K+ channels. Proc Natl Acad Sci USA 94 10461-6... 

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... 

For the purpose of determining the iron-coordination structure of nonheme iron proteins, reconstitution experiments from apoprotein and other constituents are an elegant approach which can inductively indicate the original iron structure. 

Figure 36 568.2-nm excited RR spectrum in the Fe-S stretching region for (a) natural abundance D. gigas lubredoxin and (b) of the protein reconstituted with Fe... 

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.

The EPR results for thePsaC Cysl4->Asp andPsaC Cys-51->Asp mutant proteins reconstituted with the PS-I core complex, combined with knowledge available on cysteine coordination patterns in bacterial ferredoxins containing two [4Fe 4S] clusters, as discussed below, permitted Zhao et a/. to conclude that cysteine coordination to the two iron-sulfur centers in PsaC assumes the same pattern as in bacterial ferredoxin. 

A key difference between crystalline samples and hydrated or lipid-embedded proteins can be the different degrees of molecular mobility. This aspect will impact on the utility of those solid-state NMR experiments that exploit anisotropic interactions in the magnetic field. Such aspects can, for example, play an important role if proteins reconstituted into lipid bilayers are studied at high temperatures (i.e., in the liquid crystalline phase). Here, the degree of molecular mobility may depend on the polypeptide topology in the membrane and a theoretical understanding how solid-state NMR parameters are affected by molecular motion is crucial. The basic interactions, present in a solid-state NMR experiment on spin-V2 nuclei, are introduced in the next section. 

The fact that the lack of 3,e-carotenoids in Scenedesmus and Chlamydomonas strongly impairs the accumulation of the major LHCII hut leaves some minor Chi a/b complexes unaffected indicates that in these complexes lutein can be more easily replaced with )3,/3-carotenoids (Section III.B.2). Consistent with this notion, the minor Chi a/b protein LhcbS (CP26) reconstituted with pigment mixtures lacking lutein is more stable than the major Chi a/b protein reconstituted with the same pigments (Section... 

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. 

The Mossbauer spectrum of the [4Fe-4S]- form of Dg Fd II consists of doublets 1 and 2 in a 1 3 intensity ratio (Fig. 9). When cluster conversion is carried out with 95% enriched Fe, the spectrum of the product consists of the more intense doublet 2 (132). Thus, in this case the externally supplied Fe occupies one or more of the three subsites b, which are equivalent by Mossbauer spectroscopy. Analysis of the Mossbauer spectra of the [4Fe-4S] cluster produced by dithionite reduction yielded results consistent with this picture. It was also established that the different subsites do not equilibrate at 25 °C over a time sufficient for protein reconstitution and cluster conversion. Oxidation of the [4Fe-4S] cluster with ferri-cyanide followed by examination of the product with EPR and Mossbauer spectroscopies proved transformation to the [3Fe-4S] form, thereby completing one cycle of cluster interconversion. 

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