Effect of lipid bilayer properties on the photocycle of green proteorhodopsin

The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-β-d-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli

Exploring the Interplay of Lipids and Membrane Proteins

The interplay between lipids and membrane proteins is known to affect membrane protein topology and thus have significant effect (control) on their functions. In this PhD thesis, the influence of lipids on the membrane protein function was studied using three different membrane protein models. A monotopic membrane protein, monoglucosyldiacylglyecerol synthase (MGS) from Acholeplasma laidlawii is known to induce intracellular vesicles when expressed in Escherichia coli. The mechanism leading to this unusual phenomenon was investigated by various biochemical and biophysical techniques. The results indicated a doubling of lipid synthesis in the cell, which was triggered by the selective binding of MGS to anionic lipids. Multivariate data analysis revealed a good correlation with MGS production. Furthermore, preferential anionic lipid sequestering by MGS was shown to induce a different fatty acid modeling of E. coli membranes. The roles of specific lipid binding and the probable mechanism leading to intracellular vesicle formation were also investigated. As a second model, a MGS homolog from Synechocystis sp. PCC6803 was selected. MgdA is an integral membrane protein with multiple transmembrane helices and a unique membrane topology. The influence of different type of lipids on MgdA activity was tested with different membrane fractions of Synechocystis. Results indicated a very distinct profile compared to Acholeplasma laidlawii MGS. SQDG, an anionic lipid was found to be the species of the membrane that increased the MgdA activity 7-fold whereas two other lipids (PG and PE) had only minor effects on MgdA. Additionally, a working model of MgdA for the biosynthesis and flow of sugar lipids between Synechocystis membranes was proposed. The last model system was another integral membrane protein with a distinct structure but also a different function. The envelope stress sensor, CpxA and its interaction with E. coli membranes were studied. CpxA autophosphorylation activity was found to be positively regulated by phosphatidylethanolamine and negatively by anionic lipids. In contrast, phosphorylation of CpxR by CpxA revealed to be increased with PG but inhibited by CL. Non-bilayer lipids had a negative impact on CpxA phosphotransfer activity. Taken together, these studies provide a better understanding of the significance of the interplay of lipids and model membrane proteins discussed here

Extended functional repertoire for human copper chaperones

Copper (Cu) ions are cofactors in many essential enzymes. As free Cu ions are toxic, most organisms have highly specialized Cu transport systems involving dedicated proteins. The human cytoplasmic Cu chaperone Atox1 delivers Cu to P1B-type ATPases in the Golgi network, for incorporation into Cu-dependent enzymes following the secretory path. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed CXXC motif. In addition to Atox1, the human cytoplasm also contains Cu chaperones for loading of superoxide dismutase 1 (i.e. CCS) and cytochrome c oxidase in mitochondria (i.e. Cox17). Many mechanistic aspects have been resolved with respect to how Cu ions are moved between these proteins. In addition to the primary cytoplasmic Cu chaperone function, all three cytoplasmic chaperones have been reported to have other interaction partners that are involved in signaling pathways that modulate cell growth and development. These new discoveries imply that humans have evolved a highly sophisticated network of control mechanisms that connect Cu transport with cell regulatory processes. This knowledge may eventually be exploited for future drug developments towards diseases such as cancer and neurodegenerative disorders

Fast-tumbling bicelles constructed from native Escherichia coli lipids

Solution-state NMR requires small membrane mimetic systems to allow for acquiring high-resolution data. At the same time these mimetics should faithfully mimic biological membranes. Here we characterized two novel fast-tumbling bicelle systems with lipids from two Escherichia coli strains. While strain 1 (AD93WT) contains a characteristic E. coli lipid composition, strain 2 (AD93-PE) is not capable of synthesizing the most abundant lipid in E. coli, phosphatidylethanolamine. The lipid and acyl chain compositions were characterized by P-31 and C-13 NMR. Depending on growth temperature and phase, the lipid composition varies substantially, which means that the bicelle composition can be tuned by using lipids from cells grown at different temperatures and growth phases. The hydrodynamic radii of the bicelles were determined from translational diffusion coefficients and NMR spin relaxation was measured to investigate lipid properties in the bicelles. We find that the lipid dynamics are unaffected by variations in lipid composition, suggesting that the bilayer is in a fluid phase under all conditions investigated here. Backbone glycerol carbons are the most rigid positions in all lipids, while head-group carbons and the first carbons of the acyl chain are somewhat more flexible. The flexibility increases down the acyl chain to almost unrestricted motion at its end. Carbons in double bonds and cyclopropane moieties are substantially restricted in their motional freedom. The bicelle systems characterized here are thus found to faithfully mimic E. coli inner membranes and are therefore useful for membrane interaction studies of proteins with E. coli inner membranes by solution-state NMR. (C) 2016 Elsevier B.V. All rights reserved

Heterologous overexpression of a monotopic glucosyltransferase (MGS) induces fatty acid remodeling in Escherichia coli membranes

The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellularmembraneswhen overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22 h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE)was detected to be themost affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition

Subcellular Localization of Monoglucosyldiacylglycerol Synthase in <i>Synechocystis</i> sp. PCC6803 and Its Unique Regulation by Lipid Environment

<div><p>Synthesis of monogalactosyldiacylglycerol (GalDAG) and digalactosyldiacylglycerol (GalGalDAG), the major membrane lipids in cyanobacteria, begins with production of the intermediate precursor monoglucosyldiacylglycerol (GlcDAG), by monoglucosyldiacylglycerol synthase (MGS). In <i>Synechocystis</i> sp. PCC6803 (<i>Synechocystis</i>) this activity is catalyzed by an integral membrane protein, Sll1377 or MgdA. <i>In silico</i> sequence analysis revealed that cyanobacterial homologues of MgdA are highly conserved and comprise a distinct group of lipid glycosyltransferases. Global regulation of lipid synthesis in <i>Synechocystis</i> and, more specifically, the influence of the lipid environment on MgdA activity have not yet been fully elucidated. Therefore, we purified membrane subfractions from this organism and assayed MGS activity <i>in vitro</i>, with and without different lipids and other potential effectors. Sulfoquinovosyldiacylglycerol (SQDAG) potently stimulates MgdA activity, in contrast to other enzymes of a similar nature, which are activated by phosphatidylglycerol instead. Moreover, the final products of galactolipid synthesis, GalDAG and GalGalDAG, inhibited this activity. Western blotting revealed the presence of MgdA both in plasma and thylakoid membranes, with a high specific level of the MgdA protein in the plasma membrane but highest MGS activity in the thylakoid membrane. This discrepancy in the subcellular localization of enzyme activity and protein may indicate the presence of either an unknown regulator and/or an as yet unidentified MGS-type enzyme. Furthermore, the stimulation of MgdA activity by SQDAG observed here provides a new insight into regulation of the biogenesis of both sulfolipids and galactolipids in cyanobacteria.</p></div

A simplified proposed scheme for lipid synthesis in <i>Synechocystis</i>.

<p>The semi-transparent text and arrows indicate synthesis of lipid precursors discussed in the text. The dashed lines represent putative regulatory interactions with MgdA. ACP – acyl carrier protein; G3P – glycerol-3-phosphate; CDP-DAG – CDP-diacylglycerol; Glc – glucose; Gal – galactose; PGP – phosphatidylglycerolphosphate.</p

Heterologous overexpression of a monotopic glucosyltransferase (MGS) induces fatty acid remodeling in Escherichia coli membranes :

The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22 h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~ 62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition

MGS activity in different membrane subfractions isolated from <i>Synechocystis</i>.

<p>The values shown are averages of three independent experiments, using 200 µg protein per assay. 100% activity corresponds to 23.8±1.4 pmol UDP-glucose incorporated.h⁻¹.mg total protein⁻¹. PM1– low-density plasma membrane; PM2– high-density plasma membrane; TM – thylakoid membrane. Error bars indicate standard deviations.</p