Visualizing active membrane protein complexes by electron cryotomography.

The visualization of membrane protein complexes in their natural membrane environment is a major goal in an emerging area of research termed structural cell biology. Such approaches provide important information on the spatial distribution of protein complexes in their resident cellular membrane systems and on the structural organization of multi-subunit membrane protein assemblies. We have developed a method to specifically label active membrane protein complexes in their native membrane environment with electron-dense nanoparticles coupled to an activating ligand, in order to visualize them by electron cryo-tomography. As an example, we describe here the depiction of preprotein import sites of mitochondria, formed by the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Active import sites are selectively labeled via a biotinylated, quantum dot-coupled preprotein that is arrested in translocation across the outer and inner mitochondrial membranes. Additionally, a related method is described for direct labeling of mitochondrial outer membrane proteins that does not depend on binding of a ligand.

Cryogenic single-particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single-particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in-plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in-plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

The type three secretion system (T3SS) is a bacterial weapon found in many Gram-negative bacteria. It consists out of a needle like structure called injectisome, which enables bacteria to inject effector proteins into eukaryotic cells. The injectisome is a very complex molecular machine, inserted in the inner and outer bacteria membrane, passing the periplasm with the peptidoglycan layer. We used an approach with fluorescent hybrid proteins to study the assembly order of the injectisome in Yersinia enterocolitica. First the outer membrane protein YscC was genetically fused to the red fluorescent protein mCherry. This construct was able to build fluorescent foci in the bacterial membrane by itself, without any other components of the T3SS. Then we engineered fusions between the green fluorescent protein EGFP and the inner membrane protein YscD, the putative C-ring protein YscQ or the ATPase YscN. All three constructs showed fluorescent foci at the bacterial membrane. Comparison of the different EGFP constructs with the YscC-mCherry construct in double mutants showed that the proteins co-localize. Thus we considered the foci to be a read out for injectisome assembly. By combining the EGFP constructs with different deletion mutants we found that the assembly occurs from the outside to the inside. Starting with the outer membrane protein YscC to the inner membrane. Then the ATPase and C-ring assemble together and finally the needle is formed. 
\nDifferent single particle structures of T3SS, needle complexes, purified from Shigella flexneri and Salmonella enterica are available. But in the course of purification the inner membrane export apparatus, the ATPase complex and the C-ring were lost. As well no such complex has been purified from Y. enterocolitica. Thus we investigated the Y. enterocolitica injectisomes in situ by cryo electron tomography (cryo-ET). Unfortunately, the 1-?m diameter of Y. enterocolitica is too large to obtain optimal resolution with cryo-ET. Thus we engineered a minD mutant that forms so called minicells, due to asymmetric septum placement. We collected tomograms of particles from wild type and minicells and constructed an average structure with a resolution of 3.7 nm. In addition the 6 nm resolution in situ structure of the injectisome of S. flexneri was made. We saw significant stretching of the in situ structure compared to the isolated particles. Moreover we saw flexibility of the basal body. We can only speculate that such flexibility might increase the stability of the structure and protect it of mechanical forces. In addition for the first time we could visualize a mass in cytoplasm just below the middle of the injectisome. Due to homology to the flagellum we can assume, that this is the ATPase. But to conclusively assign proteins to masses seen in the in situ structure we would need to label them.
\nA question mark in the Y. enterocolitica injectisome assembly is, how the structure can pass the peptidoglycan layer. The flagellum, which is evolutionary closely related to the injectisome, as well as other types of injectisomes have a muramidase or more specifically lytic transglycosylase (LT) encoded within their loci. No gene encoding for a LT, can be found on the pYV plasmid that encodes otherwise for the entire T3SS. Thus we tested several genomic LT for their involvement in the assembly of the T3SS. It is possible that the injectisome assembles through temporary gaps generated during the synthesis of new peptidoglycan strands. This theory is very intriguing, as the pattern of the fluorescent foci resembled closely the arrangement of the bacterial cytoskeleton protein MreB, which is assumed to be involved in placing the peptidoglycan synthesis machinery. Comparing the localization of MreB and the injectisome showed that both constructs seem to be arranged on two different helical paths. This convinced us that injectisome arrangement is not stochastic but rather controlled. To find the underlying structure responsible for this arrangement, we compared the localization of other proteins with similar arrangement as MreB. In Bacillus subtilis different membrane compositions were shown to be helically distributed. Unfortunately staining of the inner membrane is difficult in Gram-negative bacteria. Therefore we compared injectisome location with a potential bacterial lipid raft marker, which showed injectisomes do not insert into the lipid rafts. 
\nThus although our knowledge about the assembly, the structure and the function of the T3SS is improving enormously, the question of how injectisomes are localized remains to be answered.
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Ubiquinol-cytochrome-c oxidoreductase has been isolated from potato (Solanum tuberosum L.) mitochondria by cytochrome-c affinity chromatography and gel-filtration chromatography. The procedure, which up to now only proved applicable to Neurospora, yields a highly pure and active protein complex in monodisperse state. The molecular mass of the purified complex is about 650 kDa, indicating that potato cytochrome c reductase occurs as a dimer. Upon reconstitution into phospholipid membranes, the dimeric enzyme catalyzes electron transfer from a synthetic ubiquinol to equine cytochrome c with a turnover number of 50 s-1. The activity is inhibited by antimycin A and myxothiazol. A myxothiazol-insensitive and antimycin-sensitive transhydrogenation reaction, with a turnover number of 16 s-1, can be demonstrated as well. The protein complex consists of ten subunits, most of which have molecular masses similar to those of the nine-subunit fungal enzyme. Individual subunits were identified immunologically and spectral properties of b and c cytochromes were monitored. Interestingly, an additional 'core' polypeptide which is not present in other cytochrome bc1 complexes forms part of the enzyme from potato. Antibodies raised against individual polypeptides reveal that the core proteins are clearly immuno-distinguishable. The additional subunit may perform a specific function and contribute to the high molecular mass which exceeds those reported for other cytochrome-c-reductase dimers.

The production of functionally active membrane proteins (MPs) in an adequate membrane environment is a key step in structural biology. Polymer–lipid particles based on styrene and maleic acid (SMA) represent a promising type of membrane mimic, as they can extract properly folded MPs directly from their native lipid environment. However, the original SMA polymer is sensitive to acidic pH levels, which has led to the development of several modifications: SMA-EA, SMA-QA, and others. Here, we introduce a novel SMA derivative with a negatively charged taurine moiety, SMA-tau, and investigate the formation and characteristics of lipid–SMA-EA and lipid–SMA-tau membrane-mimicking particles. Our findings demonstrate that both polymers can form nanodiscs with a patch of lipid bilayer that can undergo phase transitions at temperatures close to those of the lipid bilayer membranes. Finally, we discuss the potential applications of these SMAs for NMR spectroscopy.

Membrane proteins participate in a broad range of cellular processes and represent more than 60% of drug targets. One approach to their structural analyses is mass spectrometry (MS)-based footprinting including hydrogen/deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and residue-specific chemical modification. Studying membrane proteins usually requires their isolation from the native lipid environment, after which they often become unstable. To overcome this problem, we are pursuing a novel methodology of incorporating membrane proteins into saposin A picodiscs for MS footprinting. We apply different footprinting approaches to a model membrane protein, mouse ferroportin, in picodiscs and achieve high coverage that enables the analysis of the ferroportin structure. FPOP footprinting shows extensive labeling of the extramembrane regions of ferroportin and protection at its transmembrane regions, suggesting that the membrane folding of ferroportin is maintained throughout the labeling process. In contrast, an amphipathic reagent, N-ethylmaleimide (NEM), efficiently labels cysteine residues in both extramembrane and transmembrane regions, thereby affording complementary footprinting coverage. Finally, optimization of sample treatment gives a peptic-map of ferroportin in picodiscs with 92% sequence coverage, setting the stage for HDX. These results, taken together, show that picodiscs are a new platform broadly applicable to mass spectrometry studies of membrane proteins.

Reconstitution of membrane proteins: a GPCR as an example.

Membrane proteins, lipids and detergents: not just a soap opera

Due to their unique properties, tunable size, and ability to provide a near native lipid environment, nanodiscs have found widespread use for the structural and functional studies of reconstituted membrane proteins. They have also been developed, albeit in a few applications, for therapeutic and biomedical use. For these studies and applications, it is essential to characterize the nanodisc preparations in terms of their monodispersity, size, and composition, as these can influence the properties of the membrane protein of interest. Of the many biophysical methods utilized for the study and characterization of nanodiscs, we show that analytical ultracentrifugation is able to report on sample homogeneity, shape, size, composition, and membrane protein stoichiometry or oligomerization state in a direct and simple fashion. The method is truly versatile and does not require nanodisc modification or disassembly.

Functional integrity of membrane protein rhodopsin solubilized by styrene-maleic acid copolymer

Integrating carbon nanotubes and lipid bilayer for biosensing

Lipid Bilayer Strengthens the Side Chain Interaction Network of a Membrane Protein

Membrane proteins govern many important functions in cells via dynamic oligomerization into active complexes. However, analytical methods to study their distribution and functional state in relation to the cellular structure are currently limited. Here, we introduce a technique for studying single-membrane proteins within their native context of the intact plasma membrane. SKBR3 breast cancer cells were grown on silicon microchips with thin silicon nitride windows. The cells were fixed, and the epidermal growth factor receptor ErbB2 was specifically labeled with quantum dot (QD) nanoparticles. For correlative fluorescence- and liquid-phase electron microscopy, we enclosed the liquid samples by chemical vapor deposited (CVD) graphene films. Depending on the local cell thickness, QD labels were imaged with a spatial resolution of 2 nm at a low electron dose. The distribution and stoichiometric assembly of ErbB2 receptors were determined at several different cellular locations, including tunneling nanotubes, where we found higher levels of homodimerization at the connecting sites. This experimental approach is applicable to a wide range of cell lines and membrane proteins and particularly suitable for studies involving both inter- and intracellular heterogeneity in protein distribution and expression.

Applications of solid-state NMR to membrane proteins