DGAT2 revealed by the immunogold technique in Arabidopsis thaliana lipid bodies associated with microtubules

The immunogold technique with anti-diacylglycerol acyltransferase 2 (DGAT2) antibody revealed inA. thaliana embryo and root meristematic cells gold particles manifesting the presence of DGAT2 in ER as wellas in lipid bodies. This being so, lipid synthesis could take place both in ER and in the lipid bodies. The presenceof microtubules around the lipid bodies was evidenced under transmission EM. Detection of tubulin around thelipid bodies using the immunogold technique with anti-a-tubulin is in agreement with the above observations.Connection of lipid bodies with microtubules was also detected by us in other plants where they probably participatedin lipid synthesis. A similar phenomenon may take place in A. thaliana.The immunogold technique with anti-diacylglycerol acyltransferase 2 (DGAT2) antibody revealed inA. thaliana embryo and root meristematic cells gold particles manifesting the presence of DGAT2 in ER as wellas in lipid bodies. This being so, lipid synthesis could take place both in ER and in the lipid bodies. The presenceof microtubules around the lipid bodies was evidenced under transmission EM. Detection of tubulin around thelipid bodies using the immunogold technique with anti-a-tubulin is in agreement with the above observations.Connection of lipid bodies with microtubules was also detected by us in other plants where they probably participatedin lipid synthesis. A similar phenomenon may take place in A. thaliana

Engineered β\beta-lactoglobulin produced in E-coli : purification, biophysical and structural characterisation

Functional recombinant bovine β-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained. The use of gel filtration on Sephadex G75 at the last purification step enables protein without endogenous ligand to be obtained. The physicochemical properties of recombinant β-lactoglobulin such as CD spectra, ligand binding (n, K(a), ΔH, TΔS, ΔG), chemical and thermal stability (ΔG(D), C(mid)) and crystal structure confirmed that the protein obtained is almost identical to the natural one. The substitutions of N-terminal residues did not influence the binding properties of the recombinant protein so that the lactoglobulin produced and purified according to our protocol is a good candidate for further engineering and potential use in pharmacology and medicine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12033-016-9960-z) contains supplementary material, which is available to authorized users

Complexity of seemingly simple lipid nanodiscs

Lipid nanodiscs are macromolecular assemblies, where a scaffold protein is wrapped around a nanosized disc of a lipid bilayer, thus protecting the hydrocarbon chains at the disc edges from unfavorable interactions with water. These nanostructures have numerous applications in, e.g., nanotechnology and pharmaceutics, and in investigations of membrane proteins. Here, we present results based on atomistic molecular dynamics simulations combined with electron paramagnetic spectroscopy measurements on the structure and dynamics of lipids in single-component nanodiscs. Our data highlight the existence of three distinctly different lipid fractions: central lipids residing in the center of a nanodisc, boundary lipids in direct contact with a scaffold protein, and intermediate lipids between these two regions. The central lipids are highly ordered and characterized by slow diffusion. In this part of the nanodisc, the membrane is the thickest and characterized by a gel-like or liquid-ordered phase, having features common to cholesterol-rich membranes. The boundary lipids in direct contact with the scaffold protein turned out to be less ordered and characterized by faster diffusion, and they remained in the liquid-disordered phase even at temperatures that were somewhat below the main phase transition temperature (Tm). The enthalpies associated with the central-boundary and central-intermediate transitions were similar to those observed for lipids going through the main phase transition. Overall, the study reveals lipid nanodiscs to be characterized by a complex internal structure, which is expected to influence membrane proteins placed in nanodiscs.Peer reviewe

Gγ and Gα Identity Dictate a G-Protein Heterotrimer Plasma Membrane Targeting

Heterotrimeric G-proteins along with G-protein-coupled receptors (GPCRs) regulate many biochemical functions by relaying the information from the plasma membrane to the inside of the cell. The lipid modifications of Gα and Gγ subunits, together with the charged regions on the membrane interaction surface, provide a peculiar pattern for various heterotrimeric complexes. In a previous study, we found that Gαs and Gαi3 prefer different types of membrane-anchor and subclass-specific lipid domains. In the present report, we examine the role of distinct Gγ subunits in the membrane localization and spatiotemporal dynamics of Gαs and Gαi3 heterotrimers. We characterized lateral diffusion and G-protein subunit interactions in living cells using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM), respectively. The interaction of Gγ subunits with specific lipids was confirmed, and thus the modulation of heterotrimeric G-protein localization. However, the Gα subunit also modulates trimer localization, and so the membrane distribution of heterotrimeric G-proteins is not dependent on Gγ only

Lipotubuloids - Structure and Function

Rozdział 17 książki: Advances in Selected Plant Physiology Aspects Edited by Dr. Giuseppe MontanaroThis work was realized and financed by National Committee of Scientific Research, grant No. NN 303 35 9035

Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP) protein from Escherichia coli

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically