Octyl Glucoside Research Articles

Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes.

Membrane protein characterization is consistently hampered by challenges with expression, purification, and solubilization. Among several biophysical techniques employed for their characterization, native-mass spectrometry (MS) has emerged as a powerful tool for the analysis of membrane proteins and complexes. Here, two MS platforms, the FT-ICR and Q-ToF, have been explored to analyze the homotetrameric water channel protein, AquaporinZ (AqpZ), under non-denaturing conditions. This 97 kDa membrane protein complex can be readily liberated from the octylglucoside (OG) detergent micelle under a range of instrument conditions on both MS platforms. Increasing the applied collision energy of the FT-ICR collision cell yielded varying degrees of tetramer (97 kDa) liberation from the OG micelles, as well as dissociation into the trimeric (72 kDa) and monomeric (24 kDa) substituents. Tandem-MS on the Q-ToF yielded higher intensity tetramer signal and, depending on the m/z region selected, the observed monomer signal varied in intensity. Precursor ion selection of an m/z range above the expected protein signal distribution, followed by mild collisional activation, is able to efficiently liberate AqpZ with a high S/N ratio. The tetrameric charge state distribution obtained on both instruments demonstrated superpositioning of multiple proteoforms due to varying degrees of N-terminal formylation. Graphical Abstract ᅟ.

Improved octyl glucoside synthesis using immobilized β-glucosidase on PA-M with reduced glucose surplus inhibition

A β-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (V0) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219 mM and 116 mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336 h of reaction, the OG yield and the conversion rate of glucose reached 134 mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively.

Understanding phospholipid function: Why are there so many lipids?

In the 1970s, phospholipids were still considered mere building blocks of the membrane lipid bilayer, but the subsequent realization that phospholipids could also serve as second messengers brought new interest to the field. My own passion for the unique amphipathic properties of lipids led me to seek other, non-signaling functions for phospholipids, particularly in their interactions with membrane proteins. This seemed to be the last frontier in protein chemistry and enzymology to be conquered. I was fortunate to find my way to Eugene Kennedy's laboratory, where both membrane proteins and phospholipids were the foci of study, thus providing a jumping-off point for advancing our fundamental understanding of lipid synthesis, membrane protein biosynthesis, phospholipid and membrane protein trafficking, and the cellular roles of phospholipids. After purifying and characterizing enzymes of phospholipid biosynthesis in Escherichia coli and cloning of several of the genes encoding these enzymes in E. coli and Saccharomyces cerevisiae, I was in a position to alter phospholipid composition in a systematic manner during the cell cycle in these microorganisms. My group was able to establish, contrary to common assumption (derived from the fact that membrane proteins retain activity in detergent extracts) that phospholipid environment is a strong determining factor in the function of membrane proteins. We showed that molecular genetic alterations in membrane lipid composition result in many phenotypes, and uncovered direct lipid-protein interactions that govern dynamic structural and functional properties of membrane proteins. Here I present my personal "reflections" on how our understanding of phospholipid functions has evolved.

Membrane insertion of F0 c subunit of F0F1 ATPase depends on glycolipozyme MPIase and is stimulated by YidC

The F0 c subunit of F0F1 ATPase (F0-c) possesses two membrane-spanning stretches with N- and C-termini exposed to the periplasmic (extracellular) side of the cytoplasmic membrane of E. coli. Although F0-c insertion has been extensively analyzed in vitro by means of protease protection assaying, it is unclear whether such assays allow elucidation of the insertion process faithfully, since the membrane-protected fragment, an index of membrane insertion, is a full-length polypeptide of F0-c, which is the same as the protease-resistant conformation without membrane insertion. We found that the protease-resistant conformation could be discriminated from membrane-insertion by including octyl glucoside on protease digestion. By means of this system, we found that F0-c insertion depends on MPIase, a glycolipozyme involved in membrane insertion, and is stimulated by YidC. In addition, we found that acidic phospholipids PG and CL transform F0-c into a protease-resistant form, while MPIase prevents the acquisition of such a protease-resistant conformation.

Catalytic properties of microporous zeolites in the synthesis of octyl glucoside from D-glucose with 1-octanol by single-step direct glucosidation

Catalytic properties of various microporous zeolites consisted of different acidic properties and pore topologies were studied in the synthesis of octyl glucoside from D-glucose with 1-octanol by single-step direct glucosidation. The influences of acidic properties and pore topologies of the zeolite catalysts were evaluated relating to the conversion of glucose and selectivities of octyl glucosides. The octyl glucosides could be synthesized conveniently by the single-step direct glucosidation through aging of reactants without further pre-treatment or additional supply of reactant. The reusability of the zeolite catalyst was evaluated to the used zeolite. The high conversion of D-glucose was obtained on H+ ion exchanged FAU (H-FAU) zeolite which has a mild acid strength. The conversion and yield were improved with increasing of acid site amount of the zeolite catalysts. H-FAU zeolite catalysts exhibited high octyl glucopyranoside selectivity owing to relatively a large pore cavity and a high concentration of mild acid sites. The selectivities of the octyl glucoside isomers were mainly depended on the differences of pore structure and concentration of acid sites of the zeolite catalysts. The zeolite used in the reaction was able to reuse through the regeneration process.

Insights into the Stability of GPCRs in Detergent Micelles

G-protein-coupled receptors (GPCRs) are highly dynamic membrane proteins and therefore optimization of detergents for solubilization and purification of GPCRs is an arduous task for extraction from cell membranes. The structural dynamics of GPCRs in detergents will provide key insights into the thermodynamic basis of GPCR stability in detergents. Here we report the structural insights into the stability of adenosine receptor A2AR in various detergent systems with varying tail length. We performed microseconds of atomistic molecular dynamics simulations of the A2AR in the inactive and active-like conformational states, in detergent micelles of DDM (Dodecylmaltoside), DM (Decylmaltoside), NG (Nonylglucoside), and OG (Octylglucoside). The calculated stability of the A2AR in these four detergents correlates well with the experimentally measured stability. Further analysis showed that the A2AR in DDM is less flexible and less dynamic compared to the same receptor in short tail detergents such as NG, and OG micelles. The DDM detergent preserves the helicity of the transmembrane helices in A2AR compared to other short chain detergents. NG and OG systems with shorter alkyl tails lead to unraveling of protein structure and abnormal insertion of detergent into the protein that denatures the functional conformation. We find that longer hydrophobic detergent DDM simulation more closely resembles the bilayer simulations in terms of energetics and interaction dynamics, and the insights gained from this study can be used to optimize detergents prior to experiments for GPCRs solubilization.

Dimeric peptides with three different linkers self-assemble with phospholipids to form peptide nanodiscs that stabilize membrane proteins

Three dimers of the amphipathic α-helical peptide 18A have been synthesized with different interhelical linkers inserted between the two copies of 18A. The dimeric peptides were denoted 'beltides' where Beltide-1 refers to the 18A-dimer without a linker, Beltide-2 is the 18A-dimer with proline (Pro) as a linker and Beltide-3 is the 18A-dimer linked by two glycines (Gly-Gly). The self-assembly of the beltides with the phospholipid DMPC was studied with and without the incorporated membrane protein bacteriorhodopsin (bR) through a combination of coarse-grained MD simulations, size-exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, small-angle scattering (SAS), static light scattering (SLS) and UV-Vis spectroscopy. For all three beltides, MD and combined small-angle X-ray and -neutron scattering were consistent with a disc structure composed by a phospholipid bilayer surrounded by a belt of peptides and with a total disc diameter of approximately 10 nm. CD confirmed that all three beltides were α-helical in the free form and with DMPC. However, as shown by SEC the different interhelical linkers clearly led to different properties of the beltides. Beltide-3, with the Gly-Gly linker, was very adaptable such that peptide nanodiscs could be formed for a broad range of different peptide to lipid stoichiometries and therefore also possible disc-sizes. On the other hand, both Beltide-2 with the Pro linker and Beltide-1 without a linker were less adaptable and would only form discs of certain peptide to lipid stoichiometries. SLS revealed that the structural stability of the formed peptide nanodiscs was also highly affected by the linkers and it was found that Beltide-1 gave more stable discs than the other two beltides. With respect to membrane protein stabilization, each of the three beltides in combination with DMPC stabilizes the seven-helix transmembrane protein bacteriorhodopsin significantly better than the detergent octyl glucoside, but no significant difference was observed between the three beltides. We conclude that adaptability, size, and structural stability can be tuned by changing the interhelical linker while maintaining the properties of the discs with respect to membrane protein stabilization.

What Vesicle Leakage Reveals about Antimicrobial Activity (and What It Doesn't)

The mode of action of antimicrobial peptides and their mimics is often assessed by vesicle leakage experiments, and most of these compounds are believed to act by membrane permeabilization. This work aims at improving the interpretation of vesicle leakage data in general, and at understanding and optimizing the fungicidal activity of nylon-3 polymers. We have studied the membrane permeabilizing properties of the cationic homopolymer poly-NM (Liu, R et al. JACS, 2013, 135, 5270), which displays significant antifungal activity, and two related cationic/hydrophobic binary copolymers using the lifetime-based leakage assay of calcein-loaded vesicles. We compared the results with biological activities against Candida albicans. Poly-NM induces all-or-none leakage of vesicles that are made from yeast polar lipid extract (YPLE), at the polymer's MIC against C. albicans (3 μg/mL). At this and higher concentrations, leakage requires a lag time but then proceeds to 100%. Concerted activity tests imply that the activity of the polymer does not involve detergent-like effects. Both vesicle leakage and antimicrobial activity against C. albicans spheroplasts are independent of the presence of a detergent, octyl glucoside (OG). Negligible activity is found against zwitterionic vesicles or red blood cells. All these characteristics provide a consistent, detailed picture of membrane leakage induced by electrostatic lipid clustering. The cationic/hydrophobic binary copolymer 40:60 MM:CO shows a fundamentally different pattern. Vesicle leakage is transient (limited to <100%) and graded, unspecific between zwitterionic and YPLE vesicles, additive with detergent action, and correlates poorly with biological activity. This activity profile suggests action by membrane asymmetry stress. We conclude that comprehensive leakage experiments can identify the mode of action for model membrane disruption, and we hypothesize that the correlation between vesicle leakage and antimicrobial activity is good for some types of membrane leakage but not for others.

Correlating antimicrobial activity and model membrane leakage induced by nylon-3 polymers and detergents.

Most antimicrobial peptides act upon target microorganisms by permeabilizing their membranes. The mode of action is often assessed by vesicle leakage experiments that use model membranes, with the assumption that biological activity correlates with the permeabilization of the lipid bilayer. The current work aims to extend the interpretation of vesicle leakage results and examine the correlation between vesicle leakage and antimicrobial activity. To this end, we used a lifetime-based leakage assay with calcein-loaded vesicles to study the membrane permeabilizing properties of a novel antifungal polymer poly-NM, two of its analogs, and a series of detergents. In conjunction, the biological activities of these compounds against Candida albicans were assessed and correlated with data from vesicle leakage. Poly-NM induces all-or-none leakage in polar yeast lipid vesicles at the polymer's MIC, 3 μg mL(-1). At this and higher concentrations, complete leakage after an initial lag time was observed. Concerted activity tests imply that this polymer acts independently of the detergent octyl glucoside (OG) for both vesicle leakage and activity against C. albicans spheroplasts. In addition, poly-NM was found to have negligible activity against zwitterionic vesicles and red blood cells. Our results provide a consistent, detailed picture of the mode of action of poly-NM: this polymer induces membrane leakage by electrostatic lipid clustering. In contrast, poly-MM:CO, a nylon-3 polymer comprised of both cationic and hydrophobic segments, seems to act by a different mechanism that involves membrane asymmetry stress. Vesicle leakage for this polymer is transient (limited to <100%) and graded, non-specific among zwitterionic and polar yeast lipid vesicles, additive with detergent action, and correlates poorly with biological activity. Based on these results, we conclude that comprehensive leakage experiments can provide a detailed description of the mode of action of membrane permeabilizing compounds. Without this thorough approach, it would have been logical to assume that the two nylon-3 polymers we examined act via similar mechanisms; it is surprising that their mechanisms are so distinct. Some, but not all mechanisms of vesicle permeabilization allow for antimicrobial activity.

Octyl glucoside derivatives: A tool against metal pollutants

Derivatives of octyl glucoside modified by the introduction of a chemical function (carboxylic acid, hydroxamic acid) able to enhance the complexing properties of the surfactants toward metallic cations have been used as flotation collectors to remove various metallic cations from a water phase. Flotation experiments at the laboratory scale were performed on Fe(III), Cu(II), Cr(III), Cd(II), Zn(II), Ni(II) and As(III) aqueous solutions. The most interesting results were obtained in the extraction of Fe(III) and Cu(II). Interfacial properties were studied, and the effect of the polar head structure and anomeric configuration was analyzed. Structural variations have an influence on the efficiency and the foaming ability of these compounds. The introduction of a chelating functionality preserves the interfacial performances of these surfactants. Complexes equilibrium constants and species distribution diagrams for Fe(III) and the best flotation agents 2a and 2b were determined from multi-wavelength spectrophotometric pH titration. Both compounds showed analogous values for log β1, log β2 and log β3. The bidentanted Fe(III)-hydroxamate species (2:1 surfactant-metal ratio) reached a maximum concentration in the pH range of 5.5-6.5. At this pH, the best results in the flotation experiments were obtained for both compounds.

Amphiphilic, hydrophilic, or hydrophobic synthetic bacteriochlorins in biohybrid light-harvesting architectures: consideration of molecular designs.

Biohybrid light-harvesting architectures can be constructed that employ native-like bacterial photosynthetic antenna peptides as a scaffold to which synthetic chromophores are attached to augment overall spectral coverage. Synthetic bacteriochlorins are attractive to enhance capture of solar radiation in the photon-rich near-infrared spectral region. The effect of the polarity of the bacteriochlorin substituents on the antenna self-assembly process was explored by the preparation of a bacteriochlorin-peptide conjugate using a synthetic amphiphilic bacteriochlorin (B1) to complement prior studies using hydrophilic (B2, four carboxylic acids) or hydrophobic (B3) bacteriochlorins. The amphiphilic bioconjugatable bacteriochlorin B1 with a polar ammonium-terminated tail was synthesized by sequential Pd-mediated reactions of a 3,13-dibromo-5-methoxybacteriochlorin. Each bacteriochlorin bears a maleimido-terminated tether for attachment to a cysteine-containing analog of the Rhodobacter sphaeroides antenna β-peptide to give conjugates β-B1, β-B2, and β-B3. Given the hydrophobic nature of the β-peptide, the polarity of B1 and B2 facilitated purification of the respective conjugate compared to the hydrophobic B3. Bacteriochlorophyll a (BChl a) associates with each conjugate in aqueous micellar media to form a dyad containing two β-peptides, two covalently attached synthetic bacteriochlorins, and a datively bonded BChl-a pair, albeit to a limited extent for β-B2. The reversible assembly/disassembly of dyad (β-B2/BChl)2 was examined in aqueous detergent (octyl glucoside) solution by temperature variation (15-35°C). The energy-transfer efficiency from the synthetic bacteriochlorin to the BChl-a dimer was found to be 0.85 for (β-B1/BChl)2, 0.40 for (β-B2/BChl)2, and 0.85 for (β-B3/BChl)2. Thus, in terms of handling, assembly and energy-transfer efficiency taken together, the amphiphilic design examined herein is more attractive than the prior hydrophilic or hydrophobic designs.

Additive and Synergistic Membrane Permeabilization by Antimicrobial (Lipo)Peptides and Detergents

Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index of unity, and the extent and mechanism of leakage is measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Good additivity was found for the concerted action of magainin 2, the fungicidal lipopeptide class of surfactins from Bacillus subtilis QST713, and the detergent octyl glucoside, respectively, with the detergent C12EO8. Synergistic or superadditive action was observed for fengycins from B. subtilis, as well as the detergent CHAPS, when combined with C12EO8. The results illustrate two mechanisms of synergistic action: First, maximal leakage requires an optimum degree of heterogeneity in the system that may be achieved by mixing a graded with an all-or-none permeabilizer. (The optimal perturbation should be focused to certain defect structures, yet not to the extent that some vesicles are not affected at all.) Second, a cosurfactant may enhance the bioavailability of a poorly soluble peptide. The results are important for understanding the concerted action of membrane-permeabilizing compounds in biology as well as for optimizing formulations of such antimicrobials for medical applications or crop protection.

Optimized Methods for Targeted Peptide-Based Quantification of Human Uridine 5′-Diphosphate-Glucuronosyltransferases in Biological Specimens Using Liquid Chromatography–Tandem Mass Spectrometry

The aim of this study was to optimize methods for quantifying 13 uridine 5'-diphosphate-glucuronosyltransferase (UGT) isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17) in human liver, intestinal, and kidney microsomes, and in recombinant human UGT-expressing insect cell membranes (rhUGTs) by targeted peptide-based quantification using liquid chromatography-tandem mass spectrometry. Production of targeted peptides was compared by combining three denaturing agents (urea, sodium deoxycholate, and octyl glucoside) and three denaturing temperatures (37°C, 60°C, and 95°C) followed by tryptic digestion for 2-20 hours. Denaturing conditions and digestion times yielding high production efficiency varied markedly among isoforms and specimens, indicating the importance of specific optimization. Each UGT isoform was quantified using the methods found to be optimal. The expression of 10 (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17), 6 (1A1, 1A3, 1A4, 1A10, 2B7, and 2B17), and 3 (1A6, 1A9, and 2B7) isoforms was detected in human liver, intestinal, and kidney microsomes, respectively, and levels were reproducible using multiple protocols. All isoforms were quantified in rhUGTs. Determining the levels of UGTs in human tissue specimens and those in rhUGTs is important for estimating the contribution of glucuronidation to body clearance based on in vitro-in vivo extrapolation.

Understanding Colicin N Import into Gram Negative Bacterial Cells using Small Angle Neutron Scattering

Escherichia coli (E. coli) are gram-negative bacteria commonly found in human guts but which can cause food poisoning or severe systemic infections such as meningitis. Unlike other bacteria, gram-negative cells have an additional asymmetric outer membrane (OM) which consists of tightly-packed lipopolysaccharides in the outer leaflet of membrane and phospholipids on the inside. The OM is a selective permeability barrier and protects E. coli from antibiotics and bile acids. The occurrence of antibiotic-resistant bacteria is becoming increasingly serious and owing to the tightly-packed OM gram-negative bacteria can show even higher antibiotic resistance. In the search for novel effective antibiotics, Colicin N (ColN) is a promising model. ColN is a bacterial toxin produced and secreted by E. coli in time of stress. It translocates across the OM of target cells and kills via a voltage dependent channel in the inner membrane. ColN exploits outer membrane protein F (OmpF) as both a receptor and translocator. The pore-forming domain binds to the outside of OmpF (1, 2). Here we study the OmpF/ColN translocation complex in the presence of non-ionic detergents, where binding is driven by the translocation domain. The structure of complexes was determined by small angle neutron scattering using the SANS2D beam line and utilizing a contrast variation strategy with selective deuteration of proteins and Octyl Glucoside (OG). Mixing of three OG forms achieved exact contrast matches with the solvent. This enabled a low resolution structure of the translocon to be derived from ab initio modeling and revealed that the translocation and receptor-binding domains of ColN bind to the middle of the OmpF trimer.1. Baboolal, T. G. et al. Structure16, 371, (2008).2. Clifton, L. A. et al. J. Biol. Chem.287, 337,(2012).

1+1=3? Concerted Action of Membrane Permeabilizers

Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index (FICI) of unity, and the extent and mechanism of leakage are measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Concerted action was tested for the detergent C12EO8 with the detergents octyl glucoside and CHAPS, the antimicrobial peptide magainin 2, and the fungicidal lipopeptides surfactin, fengycin and iturin from Bacillus subtilis QST713, respectively. The results are discussed in terms of an optimum heterogeneity of the system that governs the leakage mechanism, extent of leakage, and additivity of action with another agent. The results are important for understanding the nature of detergent-like actions of peptides as well as for optimizing formulations of such antimicrobials for medical applications or crop protection.

Excited state proton transfer dynamics of an eminent anticancer drug, ellipticine, in octyl glucoside micelle.

Photophysics and proton transfer dynamics of an eminent anticancer drug, ellipticine (EPT), have been investigated inside a biocompatible octyl-β-D-glucoside (OBG) micellar medium using steady state and time-resolved fluorescence spectroscopic techniques. EPT exists as protonated form in aqueous solution of pH 7. When EPT molecules are encapsulated in OBG micelles, protonated form is converted to neutral form in the ground state due to the hydrophobic effect of the micellar environment. Interestingly, steady state fluorescence results indicate the existence of both neutral and protonated forms of EPT in the excited state, even though neutral molecules are selectively excited, and it is attributed to the conversion of neutral to protonated form of EPT by the excited state proton transfer (ESPT) process. A clear isoemissive point in the time-resolved area normalized emission spectra (TRANES) further supports the excited state conversion of neutral to protonated form of EPT. Notably, this kind of proton transfer dynamics is not observed in other conventional micelles, such as, SDS, Triton-X and CTAB. Therefore, the observed ESPT dynamics is believed to be an outcome of combined effects of the local dielectric constant felt by EPT and the local proton concentration at the OBG micellar surface.

Expression, Folding, and Proton Transport Activity of Human Uncoupling Protein-1 (UCP1) in Lipid Membranes: EVIDENCE FOR ASSOCIATED FUNCTIONAL FORMS

Uncoupling protein-1 (UCP1) is abundantly expressed in the mitochondrial inner membrane of brown adipose tissues and has an important role in heat generation, mediated by its proton transport function. The structure and function of UCP1 are not fully understood, partially due to the difficulty in obtaining native-like folded proteins in vitro. In this study, using the auto-induction method, we have successfully expressed UCP1 in Escherichia coli membranes in high yield. Overexpressed UCP1 in bacterial membranes was extracted using mild detergents and reconstituted into phospholipid bilayers for biochemical studies. UCP1 was folded in octyl glucoside, as indicated by its high helical content and binding to ATP, a known UCP1 proton transport inhibitor. Reconstituted UCP1 in phospholipid vesicles also exhibited highly helical structures and proton transport that is activated by fatty acids and inhibited by purine nucleotides. Self-associated functional forms of UCP1 in lipid membranes were observed for the first time. The self-assembly of UCP1 into tetramers was unambiguously characterized by circular dichroism and fluorescence spectroscopy, analytical ultracentrifugation, and semi-native gel electrophoresis. In addition, the mitochondrial lipid cardiolipin stabilized the structure of associated UCP1 and enhanced the proton transport activity of the protein. The existence of the functional oligomeric states of UCP1 in the lipid membranes has important implications for understanding the structure and proton transport mechanism of this protein in brown adipose tissues as well as structure-function relationships of other mammalian UCPs in other tissues.

Thermal Stress Induced Aggregation of Aquaporin 0 (AQP0) and Protection by α-Crystallin via Its Chaperone Function

Aquaporin 0 (AQP0) formerly known as membrane intrinsic protein (MIP), is expressed exclusively in the lens during terminal differentiation of fiber cells. AQP0 plays an important role not only in the regulation of water content but also in cell-to-cell adhesion of the lens fiber cells. We have investigated the thermal stress-induced structural alterations of detergent (octyl glucoside)-solubilized calf lens AQP0. The results show an increase in the amount of AQP0 that aggregated as the temperature increased from 40°C to 65°C. α-Crystallin, molecular chaperone abundantly present in the eye lens, completely prevented the AQP0 aggregation at a 1∶1 (weight/weight) ratio. Since α-crystallin consists of two gene products namely αA- and αB-crystallins, we have tested the recombinant proteins on their ability to prevent thermal-stress induced AQP0 aggregation. In contrast to the general observation made with other target proteins, αA-crystallin exhibited better chaperone-like activity towards AQP0 compared to αB-crystallin. Neither post-translational modifications (glycation) nor C-terminus truncation of AQP0 have any appreciable effect on its thermal aggregation properties. α-Crystallin offers similar protection against thermal aggregation as in the case of the unmodified AQP0, suggesting that αcrystallin may bind to either intracellular loops or other residues of AQP0 that become exposed during thermal stress. Far-UV circular dichroism studies indicated a loss of αhelical structures when AQP0 was subjected to temperatures above 45°C, and the presence of α-crystallin stabilized these secondary structures. We report here, for the first time, that α-crystallin protects AQP0 from thermal aggregation. Since stress-induced structural perturbations of AQP0 may affect the integrity of the lens, presence of the molecular chaperone, α-crystallin (particularly αA-crystallin) in close proximity to the lens membrane is physiologically relevant.

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Enzyme-catalyzed synthesis of heptyl-β-glycosides: Effect of water coalescence at high temperature

Alkyl glycosides can be synthesized by glycosidases in organic media with limited amounts of water. These systems, however, limit the solubility of the sugar substrates and decrease reaction yields. Herein we report the enzymatic synthesis of heptyl-β-glycosides in heptanol catalyzed by a hyperthermophilic β-glycosidase at 90°C. Our results indicate that dispersion of water in heptanol changes with time producing coalescence of water at the bottom of the reactor, playing a key role in the reaction yield. Water-soluble substrate, enzyme and products are concentrated in the aqueous phase, according to their partition coefficients, promoting side reactions that inactivate the enzyme. Reaction yield of heptyl-β-glycosides was 35% relative to lactose, at 7% water. The increase in the water phase to 12% diminished the enzyme inactivation and increased the heptyl-β-glycosides yield to 52%. Surface-active compounds, SDS and octyl glucoside, increased water dispersion but were unable to prevent coalescence.