Binding Interactions Between α-glucans from Lactobacillus reuteri and Milk Proteins Characterised by Surface Plasmon Resonance

Interactions between milk proteins and ??-glucans at pH 4.0?C5.5 were investigated by use of surface plasmon resonance. The ??-glucans were synthesised with glucansucrase enzymes from Lactobacillus reuteri strains ATCC-55730, 180, ML1 and 121. Variations in the molecular characteristics of the ??-glucans, such as molecular weight, linkage type and degree of branching, influenced the interactions with native and denatured ??-lactoglobulin and ??-casein. The highest overall binding levels were reached with ??-(1,4) compared to ??-(1,3) linked glucans. Glucans with many ??-(1,6) linkages demonstrated the highest binding levels to ??-casein, whereas the interaction with native ??-lactoglobulin was suppressed by ??-(1,6) linkages. Glucans with a higher degree of branching generally displayed lower protein binding levels whereas a higher molecular weight resulted in increased binding to ??-casein. The interactions with ??-casein were not pH dependent, whereas binding to denatured ??-lactoglobulin was highest at pH 4.0 and binding to native ??-lactoglobulin was optimal at pH 4.5?C5.0. This study shows that molecular weight, linkage type and degree of branching of ??-glucans highly influence the binding interactions with milk proteins.     

Elimination of a misfolded folding intermediate by a single point mutation

We present an analysis of the folding behavior of the 159-residue major birch pollen allergen Bet v 1. The protein contains a water-filled channel running through it. Consequently, the protein has a hydrophobic shell, rather than a hydrophobic core. During the folding of the protein from either the urea-, pH-, or SDS-denatured state, Bet v 1 transiently populates a partially folded intermediate state. This state appears to be misfolded, since it has to unfold at least partially to fold to the native state. The misfolded intermediate is not, however, a result of the water-filled channel in Bet v 1. The intermediate completely disappears in the mutant Tyr --> Trp120, in which the channel is still present. Tyr120 appears to behave as a "negative gatekeeper" which attenuates efficient folding. The close structural homologue, the apple allergen Mal d 1, also folds without any detectable folding intermediates. However, the position of the transition state on the reaction coordinate, which is a measure of its overall compactness relative to the denatured and native states, is reduced dramatically from ca. 0.9 in Bet v 1 to around 0.5 in Mal d 1. We suggest that this large shift in the transition state structure is partly due to different local helix propensities. Given that individual mutations can have such large effects on folding, one should not a priori expect structurally homologous proteins to fold by the same mechanism.     

From lanosterol to cholesterol: structural evolution and differential effects on lipid bilayers

Cholesterol is an important molecular component of the plasma membranes of mammalian cells. Its precursor in the sterol biosynthetic pathway, lanosterol, has been argued by Konrad Bloch (Bloch, K. 1965. Science. 150:19-28; 1983. CRC Crit. Rev. Biochem. 14:47-92; 1994. Blonds in Venetian Paintings, the Nine-Banded Armadillo, and Other Essays in Biochemistry. Yale University Press, New Haven, CT.) to also be a precursor in the molecular evolution of cholesterol. We present a comparative study of the effects of cholesterol and lanosterol on molecular conformational order and phase equilibria of lipid-bilayer membranes. By using deuterium NMR spectroscopy on multilamellar lipid-sterol systems in combination with Monte Carlo simulations of microscopic models of lipid-sterol interactions, we demonstrate that the evolution in the molecular chemistry from lanosterol to cholesterol is manifested in the model lipid-sterol membranes by an increase in the ability of the sterols to promote and stabilize a particular membrane phase, the liquid-ordered phase, and to induce collective order in the acyl-chain conformations of lipid molecules. We also discuss the biological relevance of our results, in particular in the context of membrane domains and rafts.     

Understanding Detergent Effects on Lipid Membranes: A Model Study of Lysolipids

Lysolipids and fatty acids are the natural products formed by the hydrolysis of phospholipids. Lysolipids and fatty acids form micelles in solution and acts as detergents in the presence of lipid membranes. In this study, we investigate the detergent strength of a homologous series of lyso-phosphatidylcholine lipids (LPCs) on 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphatidylcholine (POPC) lipid membranes by use of isothermal titration calorimetry and vesicle fluctuation analysis. The membrane partition coefficient (K) and critical micelle concentration (cmc) are determined by isothermal titration calorimetry and found to obey an inverse proportionality relation (cmc·K ∼ 0.05-0.3). The partition coefficient and critical micelle concentration are used for the analysis of the effect of LPCs on the membrane bending rigidity. The dependency of the bending rigidity on LPC membrane coverage has been analyzed in terms of a phenomenological model based on continuum elastic theory, which yields information about the curvature-inducing properties of the LPC molecule. The results reveal: 1), an increase in the partition coefficient with increasing LPC acyl-chain length; and 2), that the degree of acyl-chain mismatch between LPC and POPC determines the magnitude of the membrane mechanical perturbation per LPC molecule in the membrane. Finally, the three-stage model describing detergent membrane interaction has been extended by a parameter D_MCI, which governs the membrane curvature stability in the detergent concentration range below the cmc-value of the LPC molecule.     

Universal behavior of membranes with sterols

Lanosterol is the biosynthetic precursor of cholesterol and ergosterol, sterols that predominate in the membranes of mammals and lower eukaryotes, respectively. These three sterols are structurally quite similar, yet their relative effects on membranes have been shown to differ. Here we study the effects of cholesterol, lanosterol, and ergosterol on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipid bilayers at room temperature. Micropipette aspiration is used to determine membrane material properties (area compressibility modulus), and information about lipid chain order (first moments) is obtained from deuterium nuclear magnetic resonance. We compare these results, along with data for membrane-bending rigidity, to explore the relationship between membrane hydrophobic thickness and elastic properties. Together, such diverse approaches demonstrate that membrane properties are affected to different degrees by these structurally distinct sterols, yet nonetheless exhibit universal behavior.     

Blocking antibodies induced by specific allergy vaccination prevent the activation of CD4+ T cells by inhibiting serum-IgE-facilitated allergen presentation.

Allergen-specific CD4+ T lymphocytes are activated at extremely low allergen concentrations in vivo as a result of serum-facilitated allergen presentation (S-FAP). It is not clear at present if specific allergy vaccination (SAV) has an effect on this mechanism. Here we show that birch allergen-specific serum-IgE facilitates the presentation of Bet v 1, the major birch pollen allergen, to Bet v 1-specific CD4+ T lymphocytes by a factor of >100. This process is CD23 mediated, could be detected in sera from the majority of birch-allergic patients, and was clearly dose dependent. S-FAP of Bet v 1 was inhibited in patients undergoing long-term birch SAV, but not by sera from patients undergoing grass SAV, indicating that birch-specific Abs are involved. This resulted in decreased proliferation and IL-4, IL-5, IL-10, and IFN-gamma production of Bet v 1-specific T cells. The inhibition was already noted after 3-9 mo of SAV and could not be solely explained by increased serum levels of birch-specific IgG4. When IgG- and IgA/IgM-containing fractions of long-term SAV sera were used to inhibit S-FAP, only IgG-containing fractions were shown to inhibit S-FAP. These results indicate that blocking IgG Abs induced by SAV inhibits the occurrence of S-FAP at very low allergen concentrations, resulting in significantly higher allergen threshold levels to obtain T cell proliferation and cytokine production and thus allergen-induced late-phase responses.     

Theory of thermal anomalies in the specific heat of lipid bilayers containing cholesterol.

A theoretical explanation of the experimentally observed characteristic thermal anomalies in the specific heat of lipid bilayers containing cholesterol is provided in terms of the phase equilibria in the phosphatidylcholine-cholesterol system. The phase equilibria are calculated via a microscopic interaction model that takes proper account of both the conformational and the crystalline degrees of freedom of the lipid acyl chains. It is shown that the characteristic double-peaked specific heat, with a narrow and a broad component, is a natural consequence of the topology of the phase diagram. Some results for the enthalpy of the mixed system are also reported. It is suggested that there is no need for invoking special mechanisms such as lipid-cholesterol complexing or formation of special interfacial regions in the bilayer in order to explain the specific-heat anomalies.     

Kinetic studies of urokinase-catalysed hydrolysis of 5-oxo-L-prolylglycyl-L-arginine 4-nitroanilide

The enzymic properties of urokinase (EC 3.4.21.31) were studied. The kinetic parameters of hydrolysis of 5-oxo-Pro-Gly-Arg-NA were determined in the pH range 5-9, at 25 degrees C and 37 degrees C. The reaction is affected by only one ionizing group of urokinase with pK 7.15 (25 degrees C) and pK 6.82 (37 degrees C). The results indicate that 5-oxo-Pro-Gly-Arg-NA is a good model substrate for studies of the conversion of plasminogen to plasmin. The Km values of the urokinase-catalysed hydrolysis of plasminogen and 5-oxo-Pro-Gly-Arg-NA are of the same order of magnitude. Plasmin catalyses the hydrolysis of 5-oxo-Pro-Gly-Arg-NA, but the Km value is several hundred times that of urokinase. Urokinase is shown not to react with good plasmin substrates, such as Bz-Arg-OEt and D-Val-Leu-Lys-NA, but is linearly competitively inhibited by 6-amino-hexanoic acid and trans-4-aminomethylcyclohexane-1-carboxylic acid.     

Steady-state kinetics of plasmin- and trypsin-catalysed hydrolysis of a number of tripeptide-p-nitroanilides.

The steady-state kinetics of plasmin- (EC 3.4.21.7) and trypsin-catalysed (EC 3.4.21.4) hydrolysis of Bz-L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA, L-Phe-Val-Arg-pNA, D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA were investigated in the pH range 6-9. The pH dependences of the kinetic parameters correspond with the effects of catalytically essential ionizations in the enzymes, except for reactions with L- and D-Phe-Val-Arg-pNA, in which protonation of the NH2-terminal alpha-amino groups (pK = 7.0) shows some inhibitory effect. The reactions of plasmin and trypsin with p-nitroanilides show kc values similar to those normally found with specific ester substrates, indicating that the deacylation steps of the reactions are rate determining.