Human serum albumin (HSA) is a major transporter for delivering several endogenous compounds including fatty acids in vivo. Even though HSA is the primary target of fatty acid binding, the effects of cationic lipid on protein stability and conformation have not been investigated. The aim of this study was to examine the interaction of human serum albumin (HSA) with helper lipids--cholesterol (Chol) and dioleoylphosphatidylethanolamine (DOPE)--and with cationic lipids--dioctadecyldimethylammonium bromide (DDAB) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), at physiological conditions, using constant protein concentration and various lipid contents. Fourier transform infrared (FTIR), circular dichroism (CD), and fluorescence spectroscopic methods were used to analyze the lipid binding mode, the binding constant, and the effects of lipid interaction on HSA stability and conformation. Structural analysis showed that cholesterol and DOPE (helper lipids) interact mainly with HSA polypeptide polar groups and via hydrophobic moieties. Hydrophobic interactions dominate the binding of cationic lipids to HSA. The number of bound lipids (n) calculated was 1.22 (cholesterol), 1.82 (DDAB), 1.76 (DOPE), and 1.56 (DOTAP). The overall binding constants estimated were KChol=2.3 (+/-0.50)x10(3) M(-1), KDDAB=8.9 (+/-0.95)x10(3) M(-1), KDOTAP=9.1 (+/-0.90)x10(3) M(-1), and KDOPE=4.7 (+/-0.70)x10(3) M(-1). HSA conformation was stabilized by cholesterol and DOPE with a slight increase of protein alpha-helical structures, while DOTAP and DDAB induced an important (alpha-->beta) transition, suggesting a partial protein unfolding.

There are several lipid binding sites on serum albumins. The aim of this study was to examine the binding of bovine serum albumin (BSA) to cholesterol (Chol), 1,2-dioleoyl-3-(trimethylammonium)propane (DOTAP), (dioctadecyldimethyl)ammonium bromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE), at physiological conditions, using constant protein concentration and various lipid contents. Fourier transform infrared (FTIR), circular dichroism (CD) and fluorescence spectroscopic methods were used to analyze the lipid binding mode, the binding constant, and the effects of lipid complexation on BSA stability and conformation. Structural analysis showed that lipids bind BSA via both hydrophilic and hydrophobic contacts with overall binding constants of K(Chol) = (1.12 +/- 0.40) x 10(3) M(-1), K(DDAB) = (1.50 +/- 0.50) x 10(3) M(-1), K(DOTAP) = (2.45 +/- 0.80) x 10(3) M(-1), and K(DOPE) = (1.35 +/- 0.60) x 10(3) M(-1). The numbers of bound lipid (n) were 1.1 (cholesterol), 1.28 (DDAB), 1.02 (DOPE), and 1.21 (DOTAP) in these lipid-BSA complexes. DDAB and DOTAP induced major alterations of BSA conformation, causing a partial protein unfolding, while cholesterol and DOPE stabilized protein secondary structure.

Background: Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed transesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially. Results: We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the halfinactivation time at 50°C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis. Conclusions: Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.

Complexes of cationic liposomes with DNA are promising tools to deliver genetic information into cells for gene therapy and vaccines. Electrostatic interaction is thought to be the major force in lipid–DNA interaction, while lipid-base binding and the stability of cationic lipid–DNA complexes have been the subject of more debate in recent years. The aim of this study was to examine the complexation of calf-thymus DNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant DNA concentration and various lipid contents. Fourier transform infrared (FTIR), UV-visible, circular dichroism spectroscopic methods and atomic force microscopy were used to analyse lipid-binding site, the binding constant and the effects of lipid interaction on DNA stability and conformation. Structural analysis showed a strong lipid–DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of KChol = 1.4 (±0.5) × 104 M−1, KDDAB = 2.4 (±0.80) × 104 M−1, KDOTAP = 3.1 (±0.90) × 104 M−1 and KDOPE = 1.45 (± 0.60) × 104 M−1. The order of stability of lipid–DNA complexation is DOTAP>DDAB>DOPE>Chol. Hydrophobic interactions between lipid aliphatic tails and DNA were observed. Chol and DOPE induced a partial B to A-DNA conformational transition, while a partial B to C-DNA alteration occurred for DDAB and DOTAP at high lipid concentrations. DNA aggregation was observed at high lipid content.

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of KChol = 5.94 (± 0.8) × 104 M–1, KDDAB = 8.33 (± 0.90) × 105 M–1, KDOTAP = 1.05 (± 0.30) × 105 M–1 and KDOPE = 2.75 (± 0.50) × 104 M–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.

A novel gene encoding an esterase from Geobacillus thermodenitrificans strain CMB-A2 was cloned, sequenced and functionally expressed in Escherichia coli M15. Sequence analysis revealed an open reading frame of 747 bp corresponding to a polypeptide of 249 amino acid residues (named EstGtA2). After purification, a specific activity of 2.58 U mg(-1) was detected using p-NP caprylate (C8) at 50 degrees C and pH 8.0 (optimal conditions). The enzyme catalyses the hydrolysis of triglycerides (tributyrin) and a variety of p-nitrophenyl esters with different fatty acyl chain length (C4-C16). The enzyme has potential for various industrial applications since it is characterized by its activity under a wide range of pH, from 25 to 65 degrees C. Using Geobacillus stearothermophilus Est30 esterase structure as template, a model of EstGtA2 was built using ESyPred3D. Analysis of this structural model allowed identifying putative sequence features that control EstGtA2 enzymatic properties. Based on sequence properties, multiple sequence comparisons and phylogenetic analyses, this enzyme appears to belong to a new family of carboxylesterases.

Monitoring the response of patients undergoing chemotherapeutic treatments is of great importance to predict remission success, avoid adverse effects and thus, maximize the patients’ quality of life. In the case of leukemia patients treated with E. coli l-asparaginase, monitoring the immune response by the detection of specific antibodies to l-asparaginase in the serum of patients can prevent extended immune response to the drug. Here, we developed a surface plasmon resonance (SPR) biosensor to rapidly detect anti-asparaginase antibodies directly in patients’ sera, without requiring sample pretreatment or dilution. A direct assay with SPR sensing to detect anti-asparaginase antibodies exhibited a limit of detection of 500 pM and a high sensitivity range between 100 nM and 1 μM in pooled and undiluted human serum from a commercial source. While the SPR assay showed excellent reproducibility (12% RSD) in pooled serum, challenges were encountered upon analyzing clinical samples due to high sample-to-sample variability in color and turbidity and, in all likelihood, in composition. As a result, direct detection in clinical samples was unreliable due to factors that may generally affect assays based on plasmonic detection. Addition of a secondary detection step overcame sample variability due to bulk differences in patients’ sera. By those means, the SPR biosensor was successfully applied to the analysis of clinical samples from leukemia patients undergoing asparaginase treatments and the results agreed well with the standard ELISA assay. Monitoring antibodies against drugs is common such that this type of sensing scheme could serve to monitor a plethora of immune responses in sera of patients undergoing treatment.

Bacterial lipolytic enzymes were originally classified into eight different families defined by Arpigny and Jaeger (families I-VIII). Recently, the discovery of new lipolytic enzymes allowed for extending the original classification to fourteen families (I-XIV). We previously reported that G. thermodenitrificans EstGtA2 (access no. AEN92268) belonged to a novel group of bacterial lipolytic enzymes. Here we propose a 15th family (family XV) and suggest criteria for the assignation of protein sequences to the N’ subfamily. Five selected salt bridges, hallmarks of the N’ subfamily (E3/R54, E12/R37, E66/R140, D124/K178 and D205/R220) were disrupted in EstGtA2 using a combinatorial alanine-scanning approach. A set of 14 (R/K→A) mutants was produced, including five single, three double, three triple and three quadruple mutants. Despite a high tolerance to non-conservative mutations for folding, all the alanine substitutions were destabilizing (decreasing T m by 5 to 14°C). A particular combination of four substitutions exceeded this tolerance and prevents the correct folding of EstGtA2, leading to enzyme inactivation. Although other mutants remain active at low temperatures, the accumulation of more than two mutations had a dramatic impact on EstGtA2 activity at high temperatures suggesting an important role of these conserved salt bridge-forming residues in thermostability of lipolytic enzymes from the N’ subfamily. We also identified a particular interloop salt bridge in EstGtA2 (D194/H222), located at position i -2 and i -4 residues from the catalytic Asp and His respectively which is conserved in other related bacterial lipolytic enzymes (families IV and XIII) with high tolerance to mutations and charge reversal. We investigated the role of residue identity at position 222 in controlling stability-pH dependence in EstGtA2. The introduction of a His to Arg mutation led to increase thermostability under alkaline pH. Our results suggest primary targets for optimization of EstGtA2 for specific biotechnological purposes.