The mechanisms by which lipid bilayer properties govern or influence membrane protein functions are little understood, but a liquid-crystalline state and the presence of anionic and nonbilayer (NB)-prone lipids seem important. An Escherichia coli mutant lacking the major membrane lipid phosphatidylethanolamine (NBprone) requires divalent cations for viability and cell integrity and is impaired in several membrane functions that are corrected by introduction of the "foreign" NBprone neutral glycolipid ␣-monoglucosyldiacylglycerol (MGlcDAG) synthesized by the MGlcDAG synthase from Acholeplasma laidlawii. Dependence on Mg 2؉ was reduced, and cellular yields and division malfunction were greatly improved. The increased passive membrane permeability of the mutant was not abolished, but protein-mediated osmotic stress adaptation to salts and sucrose was recovered by the presence of MGlcDAG. MGlcDAG also restored tryptophan prototrophy and active transport function of lactose permease, both critically dependent on phosphatidylethanolamine. Three mechanisms can explain the observed effects: NB-prone MGlcDAG improves the quenched lateral pressure profile across the bilayer; neutral MGlcDAG dilutes the high anionic lipid surface charge; MGlcDAG provides a neutral lipid that can hydrogen bond and/or partially ionize. The reduced dependence on Mg 2؉ and lack of correction by high monovalent salts strongly support the essential nature of the NB properties of MGlcDAG.The lipid bilayer of biological membranes acts as a permeability barrier permitting maintenance of essential ion gradients and is also the local environment for integral and peripheral membrane proteins. Important bilayer structural features are a liquid-crystalline state, an optimal length of the lipid chains, and critical fractions of anionic and nonbilayer-(NB) 1 prone lipids. Because of their small head groups the NB-prone lipids, when embedded in a bilayer, cause a curvature elastic stress of each monolayer with closer acyl chain packing (i.e. higher chain order) that changes the lateral pressure profile across the membrane (1). Increasing the proportion of NBprone lipids leads to a lower temperature for the bilayer to NB phase transition, which in many cases is fairly close to the growth temperature of organisms (2, 3). The balance of bilayer to NB-prone lipids may affect the passive membrane permeability of the bilayer (e.g. (4)), the folding and assembly of membrane proteins (5), and the function of important cellular processes such as cell division, transport, and osmotic responses. The essential character of such NB-prone lipids for cell membrane functions has been little studied.NB-prone lipids substantially affect the activity of several different types of proteins when studied in vitro. For example, a critical conformational change of transmembrane rhodopsin (6) and the activity of the interface-bound CTP:phosphocholine cytidylyltransferase (7) are modulated by variations in NBprone lipids and membrane curvature elastic stress, respectively. Likewise...

Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco-and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayerprone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayerprone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyldiacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.

Synthesis of the nonbilayer-prone ␣-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the lipid surface charge density in the membrane of Acholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC 2.4.1.157) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX 7 E catalytic motif of the CAZy family 4 of ␣-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity for these proteins. However, alMGS and spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface binding and activity is proposed for this regulatory enzyme.Lipids are the local environment for most integral and peripheral membrane proteins, which often depend on the lipids for optimal function. The large diversity of lipids and the differences in composition and properties between membranes have made it difficult to find out common features of bilayer organization and how lipids and proteins are cooperating in local processes. Lipid-synthesizing pathways have been mapped for the most common types of lipids, and several of the corresponding enzymes catalyzing these reactions have been characterized. However, when it comes to the connection between regulation of bilayer properties and enzyme structure, very little is known (1). So far, only a few lipid-synthesizing enzymes have been crystallized. Which structural properties are involved in the catalytic mechanism of these lipid enzymes, and how are the membrane properties sensed (1)?In the well characterized plasma membrane of Acholeplasma laidlawii, the lipid composition is regulated in a manner to maintain (i) lipid phase equilibria, close to a potential bilayer to nonbilayer transition, (ii) a nearly constant radius of spontaneous curvature, and (iii) a certain anionic surface charge density of the lipid bilayer. The synthesis of the major nonbilayer-prone lipid in this membrane, monoglucosyldiacylglycerol (MGlcDAG) 1 (Scheme 1, step I), plays an important role to fulfill the two first points above but also the third, since it is strongly regulated by negatively charged lipids (e.g. the major in vivo lipid phosphatidylglycerol (PG)) (2). MGlcDAG is consecutively processed into...

In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) ␣-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC 2.4.1.157) and the (ii) ␣-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC 2.4.1.208), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N-and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N-and (catalytic) C-domains of these structurally similar membrane-associated enzymes.

In the ultra-high temperature (UHT) process, milk is subject to temperatures above 135 °C for few seconds giving a product with a shelf-life of several months. The raw milk quality, UHT process and storage conditions affect the stability. In this study, the stability of UHT milk produced in an indirect system was evaluated by studying changes in taste, colour, fat separation, fat adhesion to the package, sedimentation, gelation, heat coagulation time, pH and ethanol stability during storage for up to one year at different temperatures. UHT milk stored at 4 and 20 °C had the longest shelf-life of 34–36 weeks, limited by sediment formation. Storage at 30 and 37 °C considerably decreased the shelf-life of UHT milk to 16–20 weeks, whereby changes in sediment formation, taste and colour were the limiting factors. Our results suggest that the changes observed at the different storage temperatures can be explained by different known mechanisms.