Exploring the active site cavity of human pancreatic lipase

Within the scope of improving the efficiency of pancreatic enzyme replacement therapy in cystic fibrosis, the feasibility of shifting the pH-activity profile of pancreatic lipase toward acidic values was investigated by site specific mutagenesis in different regions of the catalytic cavity. We have shown that introducing a negative charge close to the catalytic histidine induced a shift of the pH optimum toward acidic values but strongly reduced the lipase activity. On the other hand, a negative charge in the entrance of the catalytic cleft gives rise to a lipase with improved properties and twice more active than the native enzyme at acidic pH.     

Interaction of sea cucumber saponins with multilamellar liposomes

The effect of three sea cucumber saponins, echinoside A, bivittoside D and holothurin A, on multilamellar liposomes was investigated. An ideal osmotic behavior of liposomes was described as a linear relationship between the reciprocal $\text{3}\text{2}\text{s}$ power of absorbance at 450 nm and the osmotic gradient across the membrane. Sea cucumber saponins at concentrations below critical micelle concentration (CMC) disturbed this linear relationship in liposomes composed of egg phosphatidylcholine, phosphatidic acid and cholesterol. Cholesterol-free liposomes were not susceptible to these saponins. Results of optical measurements were consistent with those of transmission electron microscopy, which showed saponin-induced changes in liposomal structure. The lytic activity of sea cucumber saponins on liposomes depended on their chemical structure.These results suggest that sea cucumber saponins as monomers can interact with liposomes and that cholesterol serve as a principal binding site for the sea cucumber saponins.     

Alpha casein micelles show not only molecular chaperone-like aggregation inhibition properties but also protein refolding activity from the denatured state

Casein micelles are a major component of milk proteins. It is well known that casein micelles show chaperone-like activity such as inhibition of protein aggregation and stabilization of proteins. In this study, it was revealed that casein micelles also possess a high refolding activity for denatured proteins. A buffer containing caseins exhibited higher refolding activity for denatured bovine carbonic anhydrase than buffers including other proteins. In particular, a buffer containing α-casein showed about a twofold higher refolding activity compared with absence of α-casein. Casein properties of surface hydrophobicity, a flexible structure and assembly formation are thought to contribute to this high refolding activity. Our results indicate that casein micelles stabilize milk proteins by both chaperone-like activity and refolding properties.     

Mechanism by which the amyloid-like fibrils of a beta 2-microglobulin fragment are induced by fluorine-substituted alcohols

Although the formation of an alpha-helix or partial unfolding of proteins has been suggested to be important for amyloid fibrils to form in alcohols, the exact mechanism involved remains elusive. To obtain further insight into the development of amyloid fibrils, we used a 22-residue peptide, K3, corresponding to Ser20 to Lys41 of intact beta2-microglobulin. Although K3 formed an alpha-helix at high concentrations of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in 10 mM HCl (pH approximately 2), the helical content was not high, indicating a low preference to do so. The partly alpha-helical conformation was converted with time into a highly ordered beta-sheet with a fibrillar morphology as revealed by atomic force microscopy. Importantly, the TFE and HFIP-induced fibrillation exhibited a concentration dependence with a maximum at approximately 20 and approximately 10% (v/v), respectively, slightly below the concentrations at which these alcohols form dynamic clusters. Focusing on the similarity of the effects of alcohol on proteins with those of sodium dodecyl sulfate (SDS), we examined the effects of SDS on K3. SDS also induced fibrils to form with a maximum at approximately 4 mM, slightly below the critical micelle concentration. These results indicate that, with an increase in the concentration of hydrophobic cosolvent (TFE, HFIP, or SDS), a delicate balance of decreasing hydrophobic interactions and increasing polar interactions (i.e. H-bonds) in and between peptides leads to the formation of ordered fibrils with a bell-shaped concentration dependence.     

Ciprofloxacin interactions with bacterial protein OmpF: Modelling of FRET from a multi-tryptophan protein trimer

The outer membrane protein F (OmpF) is known to play an important role in the uptake of fluoroquinolone antibiotics by bacteria. In this study, the degree of binding of the fluoroquinolone antibiotic ciprofloxacin to OmpF in a lipid membrane environment is quantified using a methodology based on Förster resonance energy transfer (FRET). Analysis of the fluorescence quenching of OmpF is complex as each OmpF monomer presents two tryptophans at different positions, thus sensing two different distributions of acceptors in the bilayer plane. Specific FRET formalisms were derived accounting for the different energy transfer contributions to quenching of each type of tryptophan of OmpF, allowing the recovery of upper and lower boundaries for the ciprofloxacin-OmpF binding constant (K_B). log (K_B) was found to lie in the range 3.15-3.62 or 3.58-4.00 depending on the location for the ciprofloxacin binding site assumed in the FRET modelling, closer to the centre or to the periphery of the OmpF trimer, respectively. This methodology is suitable for the analysis of FRET data obtained with similar protein systems and can be readily adapted to different geometries.     

The phospholipid fatty acids of Porphyridium purpureum cultured in the presence of triton x-100 and sodium desoxycholate

The phospholipid fatty acid composition of Porphyridium purpureum grown on a solid medium was studied in the presence of Triton X-100 (TX) and sodium desoxycholate (SDC). The most common fatty acids in PC and PE were palmitic (16:0), stearic (18:0), linoleic (18:2ω6), arachidonic (20:4ω6) and eicosapentaenoic (20:5ω3) acids, 20:4ω6 being very abundant. In PG the most common acids were 16:0, trans-hexaenoic acid (tr16:1ω3), oleic acid (18:1) and 20:4ω6. Both detergents caused an increase in the saturation of PC and, to a lesser extent, of PE. The relative amounts of short chain fatty acids increased. Both detergents increased the amounts of 16:0 and, correspondingly, decreased the amounts of 20:4ω6. In PG the amounts of both 16:0 and tr 16:1ω3 increased and the amounts of 18:0, 18:2ω6 and 20:4ω6 decreased in the presence of detergents. The changes were always greatest at the concentrations of 5–10 ppm TX or SDC. At 20 ppm the fatty acid compositions, especially with SDC, were very similar to the controls, which suggests a change in the detergent effect between 10–20 ppm. The normal PC/PE ratio was 5.6 and the (PC+ PE)/PG ratio 39.0. Both detergents caused a marked decrease in these ratios. Because the detergent effects are not linear, it seems that even very low detergent concentrations have an important influence on algae in polluted waters.     

Bcl-2 proteins and mitochondria—Specificity in membrane targeting for death

The localization and control of Bcl-2 proteins on mitochondria is essential for the intrinsic pathway of apoptosis. Anti-apoptotic Bcl-2 proteins reside on the outer mitochondrial membrane (OMM) and prevent apoptosis by inhibiting the activation of the pro-apoptotic family members Bax and Bak. The Bcl-2 subfamily of BH3-only proteins can either inhibit the anti-apoptotic proteins or directly activate Bax or Bak. How these proteins interact with each other, the mitochondrial surface and within the OMM are complex processes we are only beginning to understand. However, these interactions are fundamental for the transduction of apoptotic signals to mitochondria and the subsequent release of caspase activating factors into the cytosol. In this review we will discuss our knowledge of how Bcl-2 proteins are directed to mitochondria in the first place, a crucial but poorly understood aspect of their regulation. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

A survey of detergents for the purification of stable,active human cystic fibrosis transmembrane conductance regulator (CFTR)

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1 mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C₁₂E₈, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2–3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1 mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6–9 days in MNG or Chaps, and 12–17 days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.     

Role of the membrane interface on the conformation of the caveolin scaffolding domain: a CD and NMR study

Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of two synthetic peptides, D82-R101 and D82-I109, encompassing the caveolin scaffolding domain (D82-R101), in the presence of dodecylphosphocholine (DPC) micelles. Our data show that a stable helical conformation of the caveolin scaffolding domain in a membrane mimicking system is only obtained for the peptide including the L102-I109 hydrophobic stretch, a part of the caveolin intra-membrane domain. Through chemical shift variations, an ensemble of six residues of the D82-L109 peptide, mainly located in the V(94)TKYWFYR(101) motif were found to detect the presence of phosphatidylserine solubilized in DPC micelles. Our results constitute a first step for elucidating at a residue level the conformational properties of the central region of the caveolin-1 protein.     

Non-lipolytic and lipolytic sequence-related carboxylesterases: A comparative study of the structure–function relationships of rabbit liver esterase 1 and bovine pancreatic bile-salt-activated lipase

To differentiate esterases from lipases at the structure–function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116–123 in bBAL is deleted in rLE. This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase–lipid adsorption complex at interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site.     

Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca²⁺- and Mg²⁺-ATPase activities of CF1 deficient in its inhibitory ε subunit (CF1-ε) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca²⁺-ATPase activity by CTAB is irreversible. The Ca²⁺-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg²⁺-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-ε or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-ε from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the α/β heterohexamer.     

Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins

Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC–MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC–MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD.     

Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.     

Study of water vapor and surfactant absorption by lipid model systems using the quartz crystal microbalance

Skin is constantly exposed to surfactants which compromise the essential barrier function of normal healthy skin. To model the interactions of surfactants with the barrier lipids of the stratum corneum (SC), it is essential to develop in vitro and in vivo quantitative measurement methods to predict, evaluate, and demonstrate the effect of the different surfactant chemistries and technologies on skin. In the current work, in vitro water vapor uptake and surfactant absorption onto skin lipid model films were quantitatively studied using a technique based on the piezoelectric effect, the quartz crystal microbalance (QCM). This approach is straightforward and reliable in providing subtle surface/interface related mass change information with high resolution and sensitivity. The results show that barrier properties of the lipid model system may be damaged by surfactant absorption, as well as by long-term water exposure due to alterations to the lipid film structure. Surfactant absorption is found to be concentration dependent even beyond its critical micelle concentration (CMC). QCM results for different surfactant systems are consistent with reported clinical data in showing that clinically milder surfactants (SLES) do not perturb the film as much as clinically harsh surfactants (SDS).     

Kinetic analysis of the polymerization and depolymerization of β2-microglobulin-related amyloid fibrils in vitro

β₂-Microglobulin-related (Aβ₂M) amyloidosis is a serious complication in patients on long-term dialysis, and partial unfolding of β₂-microglobulin (β₂-m) is believed to be prerequisite to its assembly into Aβ₂M amyloid fibrils. Many kinds of amyloid-associated molecules (e.g., apolipoprotein E (apoE), glycosaminoglycans (GAGs), proteoglycans (PGs)) may contribute to the development of Aβ₂M amyloidosis. The formation of Aβ₂M amyloid fibrils in vitro was first observed at low pH (2.0–3.0). Very recently, low concentrations of 2,2,2-trifluoroethanol (TFE) and the sub-micellar concentration of sodium dodecyl sulfate, a model for anionic phospholipids, have been reported to cause the extension of Aβ₂M amyloid fibrils at a neutral pH, inducing partial unfolding of β₂-m and stabilization of the fibrils. Moreover, apoE, GAGs and PGs were found to stabilize Aβ₂M amyloid fibrils at a neutral pH, forming a stable complex with the fibrils. Some GAGs, especially heparin enhanced the fibril extension in the presence of TFE at a neutral pH. Some PGs, especially biglycan also induced the polymerization of acid-denatured β₂-m. These findings are consistent with the hypothesis that in vivo, specific molecules that affect the conformation and stability of β₂-m and amyloid fibrils will have significant effects on the deposition of Aβ₂M amyloid fibrils.     

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-?SBD and Hsp70-?NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD.     

Interaction with membrane mimics of transmembrane fragments 16 and 17 from the human multidrug resistance ABC transporter 1 (hMRP1/ABCC1) and two of their tryptophan variants

The human multidrug resistance-associated protein 1 (hMRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily. Together with P-glycoprotein (ABCB1) and the breast cancer resistance protein (BCRP/ABCG2), hMRP1 confers resistance to a large number of structurally diverse drugs. The current topological model of hMRP1 includes two cytosolic nucleotide-binding domains and 17 putative transmembrane (TM) helices forming three membrane-spanning domains. Mutagenesis and labeling studies have shown TM16 and TM17 to be important for function. We characterized the insertion of the TM16 fragment into dodecylphosphocholine (DPC) or n-dodecyl-β-d-maltoside (DM) micelles as membrane mimics and extended our previous work on TM17 (Vincent et al., 2007, Biochim. Biophys. Acta 1768, 538). We synthesized TM16 and TM17, with the Trp residues, W1198 in TM16 and W1246 in TM17, acting as an intrinsic fluorescent probe, and TM16 and TM17 Trp variants, to probe different positions in the peptide sequence. We assessed the interaction of peptides with membrane mimics by evaluating the increase in fluorescence intensity resulting from such interactions. In all micelle-bound peptides, the tryptophan residue appeared to be located, on average, in the head group micelle region, as shown by its fluorescence spectrum. Each tryptophan residue was partially accessible to both acrylamide and the brominated acyl chains of two DM analogs, as shown by fluorescence quenching. Tryptophan fluorescence lifetimes were found to depend on the position of the tryptophan residue in the various peptides, probably reflecting differences in local structures. Far UV CD spectra showed that TM16 contained significant β-strand structures. Together with the high Trp correlation times, the presence of these structures suggests that TM16 self-association may occur at the interface. In conclusion, this experimental study suggests an interfacial location for both TM16 and TM17 in membrane mimics. In terms of overall hMRP1 structure, the experimentally demonstrated amphipathic properties of these TM are consistent with a role in the lining of an at least partly hydrophilic transport pore, as suggested by the currently accepted structural model, the final structure being modified by interaction with other TM helices.     

Natural terpenes: self-assembly and membrane partitioning

Monoterpenes (MTs) are highly hydrophobic substances present in essential oils. They cover a wide spectrum of biological effects with a membrane interaction as a common point. Here we studied the surface activity of camphor, cineole, thymol, menthol and geraniol, and their ability to reach and incorporate into model membranes affecting some features of their dynamic organization. All the MTs studied self-aggregated in water with critical micellar concentrations (CMC) between 3 and 8 microM. Their octanol-water and membrane-water partition coefficients were correlated with one another. They all penetrated in monomolecular layers of dipalmitoyl-phosphatildylcholine at the air-water interface, even at surface pressures (pi) above the equilibrium lateral pressure of bilayers; thymol exhibited the highest (61.3 mN/m) and camphor the lowest (37 mN/m) pi(cut-off) value. They affected the self-aggregation of Triton X-100, increasing its CMC from 0.16 mM in the absence of MTs up to 0.68 mM (e.g. for geraniol), and the topology of sPC vesicles, increasing its surface curvature, suggesting their location at the polar head group region of the membrane. The latter was supported by their ability to increase differentially the polarity of the membrane environment sensed by two electrochromic dyes. Dipole moment values (between 1.224 and 2.523 D) and solvation areas (between 80 and 97 A(2)) were calculated from their energy-minimized structures. The relative contribution of each experimental, theoretical and structural property to determine MTs' effects on membrane dynamics were evaluated by a principal component analysis.