Structural rearrangement of β-lactoglobulin at different oil-water interfaces and its effect on emulsion stability

Understanding the factors that control protein structure and stability at the oil-water interface continues to be a major focus to optimize the formulation of protein-stabilized emulsions. In this study, a combination of synchrotron radiation circular dichroism spectroscopy, front-face fluorescence spectroscopy, and dual polarization interferometry (DPI) was used to characterize the conformation and geometric structure of β-lactoglobulin (β-Lg) upon adsorption to two oil-water interfaces: a hexadecane-water interface and a tricaprylin-water interface. The results show that, upon adsorption to both oil-water interfaces, β-Lg went through a β-sheet to α-helix transition with a corresponding loss of its globular tertiary structure. The degree of conformational change was also a function of the oil phase polarity. The hexadecane oil induced a much higher degree of non-native α-helix compared to the tricaprylin oil. In contrast to the β-Lg conformation in solution, the non-native α-helical-rich conformation of β-Lg at the interface was resistant to further conformational change upon heating. DPI measurements suggest that β-Lg formed a thin dense layer at emulsion droplet surfaces. The effects of high temperature and the presence of salt on these β-Lg emulsions were then investigated by monitoring changes in the ζ-potential and particle size. In the absence of salt, high electrostatic repulsion meant β-Lg-stabilized emulsions were resistant to heating to 90 °C. Adding salt (120 mM NaCl) before or after heating led to emulsion flocculation due to the screening of the electrostatic repulsion between colloidal particles. This study has provided insight into the structural properties of proteins adsorbed at the oil-water interface and has implications in the formulation and production of emulsions stabilized by globular proteins.     

Vibrational sum-frequency generation at protein modified air–water interfaces: Effects of molecular structure and surface charging

Protein and surfactant modified air–water interfaces are an important model system for colloid science as many applications for example aqueous foams in food products rely on our knowledge and ability to tune molecular structures at these interfaces. That is because interfaces are a fundamental building block in the hierarchical structure of foam, where in fact the molecular level can determine properties on larger length scales. For that reason it is of great importance to increase our ability to study air–water interfaces with molecular level probes and to obtain not only information on coverage but also direct information on interfacial composition, molecular order, orientations as well as information on the charged state of an interface. Vibrational sum-frequency generation (SFG) is a powerful tool that can help to address these issues and is inherently surface sensitive. In this contribution we will review recent developments in the use of SFG for studies of biomolecules at aqueous interfaces and discuss current issues with the interpretation of SFG spectra from electrified interfaces. In order to guide interpretations from interface spectroscopy we invoke the use of complementary methods such as ellipsometry and zetapotential measurements of bulk molecules.     

Correlation between structure and stability of nitronic acids and nitronic esters

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Thermodynamic study of the influence of polyols and glucose on the thermal stability of holo-bovine α-lactalbumin

Thermal stability of bovine α-lactalbumin in buffer and dilute aqueous solutions of erythritol, xylitol, sorbitol, inositol and glucose was evaluated by fluorescence spectroscopy and circular dichroism. Results show that at the selected conditions, the transition is reversible and is well described by a two-state model. At low concentration the cosolutes do not show a structure stabilizing effect, and some of them even destabilize the protein. At higher concentration, all of them stabilize the native protein conformation; however, the extent of stabilization is lower than the effect shown with other proteins, presumably due to the lactalbumin incomplete unfolding.     

Effects of the biological backbone on stacking interactions at DNA-protein interfaces: the interplay between the backbone···π and π···π components

The (gas-phase) MP2/6-31G*(0.25) π···π stacking interactions between the five natural bases and the aromatic amino acids calculated using (truncated) monomers composed of conjugated rings and/or (extended) monomers containing the biological backbone (either the protein backbone or deoxyribose sugar) were previously compared. Although preliminary energetic results indicated that the protein backbone strengthens, while the deoxyribose sugar either strengthens or weakens, the interaction calculated using truncated models, the reasons for these effects were unknown. The present work explains these observations by dissecting the interaction energy of the extended complexes into individual backbone···π and π···π components. Our calculations reveal that the total interaction energy of the extended complex can be predicted as a sum of the backbone···π and π···π components, which indicates that the biological backbone does not significantly affect the ring system through π-polarization. Instead, we find that the backbone can indirectly affect the magnitude of the π···π contribution by changing the relative ring orientations in extended dimers compared with truncated dimers. Furthermore, the strengths of the individual backbone···π contributions are determined to be significant (up to 18 kJ mol(-1)). Therefore, the origin of the energetic change upon model extension is found to result from a balance between an additional (attractive) backbone···π component and differences in the strength of the π···π interaction. In addition, to understand the effects of the biological backbone on the stacking interactions at DNA-protein interfaces in nature, we analyzed the stacking interactions found in select DNA-protein crystal structures, and verified that an additive approach can be used to examine the strength of these interactions in biological complexes. Interestingly, although the presence of attractive backbone···π contacts is qualitatively confirmed using the quantum theory of atoms in molecules (QTAIM), QTAIM electron density analysis is unable to quantitatively predict the additive relationship of these interactions. Most importantly, this work reveals that both the backbone···π and π···π components must be carefully considered to accurately determine the overall stability of DNA-protein assemblies.     

Molecular simulation of protein adsorption and conformation at gas-liquid,liquid–liquid and solid–liquid interfaces

The adsorption of proteins at surfaces and interfaces is important in a wide range of industries. Understanding and controlling the conformation of adsorbed proteins at surfaces is critical to stability and function in many technological applications including foods and biomedical testing kits or sensors. Studying adsorbed protein conformation is difficult experimentally and so over the past few decades researchers have turned to computer simulation methods to give information at the atomic level on this important area. In this review we summarize some of the significant simulation work over the past four years at both fluid (liquid–liquid and gas–liquid interfaces) and solid–liquid interfaces. Of particular significance is the work on surfactant proteins such as fungal hydrophobins, ranspumin-2 from the túngara frog and the bacteria protein BslA. These have evolved unique structures impart very high surface-active properties to the molecules. A highlight is the elucidation of the clam-shell unhinging mechanism of ranspumin-2 adsorption to the gas–liquid interface that is responsible for its adsorption to and stabilization of the air bubbles in túngara frog foam nests.     

Polarity and structure of α-nitrocinnamic acid esters

Polarity and structure of ethyl α-nitrocinnamates have been studied by means of dipole moments method and quantum-chemical calculations. These compounds exist in the form of Z-isomers in solution, that is, nitro group and benzene ring are cis-positioned.     

Complexation between α-cyclodextrin and PEGylated-PAMAM dendrimers at low and high pH values

Poly(ethylene glycol)-grafted-poly(amido amine) (PEGylated-PAMAM) dendrimers have attracted increasing amounts of attention because of their improved stability, toxicity, and better particle drug leakage property. The complexation of α-cyclodextrin (α-CD) with grafted PEG segments on the surface of PAMAM dendrimers was elucidated by light scattering and titration calorimetry. At pH 10, complexation between α-CD and PEGylated-PAMAM occurred once α-CD was titrated into the PAMAM solution. We observed for the first time a unique phenomenon at pH 2, where no binding took place until a critical α-CD concentration (C*) of ～8.0 mM was reached. The size of the nanostructures increased from 6.7 to 57.6 nm when the α-CD concentration was increased from 0.5 to 15 mM at pH 2. The zeta potential of PEGylated-PAMAM at pH 2 was +6.7 mV. Thus, the dendrimers possessed positive charges attributed to the protonation of primary amine groups on PAMAM chains that impart electrostatic repulsive forces to the system. The morphology of the complex is expected to be different at two different pH values (2 and 10) because the former produces a clear solution and the latter forms a turbid solution with white precipitates.     

The NMR Coupling Constants,and Conformational and Structural Properties of Flexible Aldopyranosyl Rings: α- and β-D-Idopyranose

ABSTRACT

The flexible ring structures of α- and β-D-idopyranose have been investigated by conformational analysis using structures generated by MacroModel and GMMX search protocols. The lowest energy structures found during the conformer search for the ⁴ C ₁, ¹ C ₄, ^O S ₂ and the ³ S ₁ structures were then examined by AM1 and Gaussian ab initio methods at the HF/6-311G** and HF/6-31+G* levels. The B _2,5 conformer found for β-D-idopyranose at 14 kJ/mol by GMMX and 29.5 kJ/mol for α-D-idopyranose by MacroModel would not contribute to Boltzmann-averaged ¹H NMR coupling constants. The Merck MMFF force field tends to overweight the ¹ C ₄ structures, making these the lowest energy conformers for both anomers. Boltzmann-averaged coupling constants are heavily weighted by this structure in the MMFF search conformer ensemble. Averaged proton coupling constants determined using MMFF fit very well for α-D-idopyranose compared to the observed values, but fit poorly for the β-anomer. Ab initio results place the ¹ C ₄ conformer at lowest energy for the α-anomer and place the ⁴ C ₁ conformer at lowest energy for the β-anomer. The GMMX and MM3* force fields find the ⁴ C ₁ conformer to have the lowest energies for both anomers.     

Biophysical analysis of partially folded state of α-lactalbumin in the presence of cationic and anionic surfactants

The role of different types of interactions and their contribution in the stabilization of bovine α-lactalbumin (α-LA) molten globule in presence of cationic surfactant, hexadecyl trimethyl ammonium bromide (HTAB) and anionic surfactant, sodium dodecyl sulphate (SDS) have been examined using a combination of spectroscopic, light scattering and calorimetric techniques. The results correlated well with each other and were used to characterize the partially folded states of the protein both qualitatively and quantitatively. At lower concentration of the surfactants, the thermodynamic parameters obtained from UV-visible spectroscopy suggested an increased exposure of non-polar groups in HTAB while a possible restructuring of non-polar groups were indicated in SDS. The fluorescence and circular dichroism spectroscopy showed the formation of an intermediate state at various concentrations of HTAB and SDS while the lifetime measurements supported the assumption of protein-surfactant complex stability in HTAB as compared to SDS. The hydrodynamic diameter and the ζ-potential were analyzed by dynamic light scattering (DLS) which also implicated the combined influence of electrostatic and hydrophobic interactions in protein unfolding in HTAB and only hydrophobic interactions in SDS. The binding parameters for ANS obtained from isothermal titration calorimetric (ITC) measurements suggested a high stability of α-LA molten globule and the role of enthalpic and entropic contribution in the binding of ANS in HTAB. It also indicated the fragility of α-LA molten globule in SDS. The possible binding sites as well as the interactions of ANS with the partially folded protein were also studied from the thermodynamic parameters obtained from the ITC.     

Conformational stability and rotational energy barrier of RC60–C60R dimers: hyperconjugation versus steric effect

The conformational stability of 4, 4?? disubstituted HC₆₀?CC₆₀R and RC₆₀?CC₆₀R dimers were calculated at ONIOM approach (AM1:B3LYP/6-31+G**) and density functional theory (B3LYP/6-31G**). The new evidences for stability and rotational energy barriers of these dimers were obtained by natural bond orbital, natural steric and molecular orbital analyses. Based on B3LYP/6-31G** calculations, except for RC₆₀?CC₆₀R (R?=?hydrogen, tert-butyl and trimethylsilyl) where gauche is the most stable conformer, trans is a global energy minimum. The greater stability of the gauche conformer of HC₆₀?CC₆₀H over trans is the result of hyperconjugation, which dominates the instability caused by the steric effect. By increasing the size of the substituent of HC₆₀?CC₆₀R dimers, the trans becomes sterically unstable but the hyperconjugation of bulky substituents dominates (the trans is global energy minimum). The hyperconjugation stability of RC₆₀?CC₆₀R dimers dominates until R?=?iso-propyl (higher stability of trans). In the case of bulky tert-butyl and trimethylsilyl substituents, the steric energy of trans is large and overweighs the hyperconjugation effect. This favors gauche as the most stable conformer. The calculated rotational energy barrier for HC₆₀?CC₆₀R and RC₆₀?CC₆₀R dimers is less than 7.3 and more than 10?kcal/mol, respectively (depending on substitution).     

Unravelling the interaction between α-cyclodextrin with the thaumatin protein and a peptide mimic

G-protein-coupled receptors (GPCRs) are responsible for signal transduction; through these transmembrane proteins, our senses are evoked: sight, smell and taste. Thaumatin is a natural sweet-tasting protein that is 100,000 times sweeter than sucrose but its use in food products has been hampered due to a liquorice aftertaste. Thaumatin has been shown to bind to a class C GPCR and the active binding site of the thaumatin protein is known. Here, we report on the binding of a well-known food grade host: α-cyclodextrin to thaumatin. We show through a combination of one- and two-dimensional NMR experiments that α-cyclodextrin binds to aromatic residues on thaumatin with K_a = 8.5 ± 2.4 M ^? 1. We also synthesise a heptapeptide KTGDRGF that mimics the active binding site of thaumatin and show that α-cyclodextrin binds to the C-terminal solvent accessible phenylalanine residue of this peptide with K_a = 8.8 ± 3.1 M ^? 1. This indicates that α-cyclodextrin may interact with the active binding site on thaumatin, suggesting that α-cyclodextrin could be used to modify the interaction of thaumatin with GPCRs and hence its sweet-taste profile.     

Study of the structure and binding site features of FaEXPA2, an α-expansin protein involved in strawberry fruit softening

Tissue softening accompanies the ripening of many fruits and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins that have been proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Several authors have shown that FaEXPA2 is a key gene that shows an increased expression level during ripening and softening of the strawberry fruit. For this reason, FaEXPA2 is frequently used as a molecular marker of softening in strawberry fruit, and changes in its relative expression have been related to changes in fruit firmness. In this context, we previously reported that FaEXPA2 has a high accumulation rate during fruit ripening in four different strawberry cultivars; however, the molecular mechanism of FaEXPA2 or expansins in general is not yet clear. Herein, a 3D model of the FaEXPA2 protein was built by comparative modeling to understand how FaEXPA2 interacts with different cell wall components at the molecular level. First, the structure was shown to display two domains characteristic of the other expansins that were previously described. The protein-ligand interaction was evaluated by molecular dynamic (MD) simulation using four different long ligands (a cellulose fiber, two of the more important xyloglucan (XG) fibers found in strawberry (XXXG and XXFG type), and a pectin (homogalacturonic acid type)). The results showed that FaEXPA2 formed a more stable complex with cellulose than other ligands via the different residues present in the open groove surface of its two domains, while FaEXPA2 did not interact with the pectin ligand.     

Modification of aluminum alkoxides with β-ketoesters: new insights into formation,structure and stability

[Al(OⁱPr)₂(β-ketoesterate)]₂ and Al(β-ketoesterate)₃ (β-ketoesterate = methyl, ethyl, iso -propyl, tert-butyl, allyl and 2-(methacryloyloxy)ethyl acetoacetate) were prepared by reaction of [Al(OⁱPr)₃]₄ with the corresponding β-ketoesters. Al(β-ketoesterate)₃ derivatives were exclusively formed at room temperature, whereas elevated reaction temperatures, causing thermal de-oligomerization of [Al(OⁱPr)₃]₄, were necessary for the formation of [Al(OⁱPr)₂(β-ketoesterate)]₂. All compounds were characterized by NMR spectroscopy, and [Al(OⁱPr)₂(tert-butyl acetoacetate)]₂ by a single crystal structure analysis. The [Al(OⁱPr)₂(β-ketoesterate)]₂ derivatives are asymmetrically substituted dimers with one octahedrally and one tetrahedrally substituted aluminum atom, bridged by two iso -propoxo groups, whereas the Al(β-ketoesterate)₃ derivatives are monomers with octahedrally coordinated aluminum. Transesterification as a possible side reaction was only observed at elevated temperatures for Al(tert-butyl acetoacetate)₃ in the presence of liberated iso -propanol. Dedicated to David Avnir on the occasion of his 60th birthday.     

Inhibition of protein interactions: co-crystalized protein–protein interfaces are nearly as good as holo proteins in rigid-body ligand docking

Modulating protein interaction pathways may lead to the cure of many diseases. Known protein–protein inhibitors bind to large pockets on the protein–protein interface. Such large pockets are detected also in the protein–protein complexes without known inhibitors, making such complexes potentially druggable. The inhibitor-binding site is primary defined by the side chains that form the largest pocket in the protein-bound conformation. Low-resolution ligand docking shows that the success rate for the protein-bound conformation is close to the one for the ligand-bound conformation, and significantly higher than for the apo conformation. The conformational change on the protein interface upon binding to the other protein results in a pocket employed by the ligand when it binds to that interface. This proof-of-concept study suggests that rather than using computational pocket-opening procedures, one can opt for an experimentally determined structure of the target co-crystallized protein–protein complex as a starting point for drug design.     

Conformational and self-assembly studies of helix forming hexapeptides containing two α-amino isobutyric acids

Single crystal X-ray diffraction studies reveal that three hexapeptides with general formula Boc-Ile-Aib-Xx-Ile-Aib-Yy-OMe, where Xx and Yy are Leu in peptide I, Leu and Phe in peptide II, and Phe and Leu in peptide III, respectively, adopt equivalent conformations that can be described as mixed 3₁₀/α-helice with two 4→1 and two 5→1 intramolecular N-H?OC H-bonds. The peptides do not generate any helix-terminating Schellman motif despite having Aib at the penultimate position from C-terminus. In the crystalline state, the helices are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. The CD studies of the three hexapeptides in acetonitrile indicate that they are folded in well-developed 3₁₀-helical structures. NMR studies of peptide I in CDCl₃ also suggest the formation of a homogeneous 3₁₀-helical structure. The field emission scanning electron microscopic (FE-SEM) images of peptide II in the solid state reveal a non-twisted ribbon-like morphology, which is formed through lateral association of non-twisted filaments.     

Interactions between synthetic and indigenous naphthenic acids and divalent cations across oil–water interfaces: effects of addition of oil-soluble non-ionic surfactants

Interactions between naphthenic acids and divalent metal cations across model oil–alkaline water interfaces were investigated by correlating changes in dynamic interfacial tension (IFT), to plausible reaction mechanisms. The measurements were carried out by using a CAM 200 optical instrument, which is based on the pendant drop technique. The naphthenic acids used were synthesised model compounds as well as commercial acid mixtures from crude distillation and extracted acid fractions from a North Sea crude oil. The divalent cations involved Ca²⁺, Mg²⁺, Sr²⁺, and Ba²⁺, which are all common in co-produced formation water and naphthenate deposits. The results show that the dynamic IFT strongly depends on naphthenic acid structure, type of divalent cation, and the concentration of the compounds as well as the pH of the aqueous phase. Introducing divalent cations to systems involving saturated naphthenic acids caused mostly a permanent lowering of the IFT. The decline in IFT is due to electrostatic attraction forces across the interface between the cations in the aqueous phase and the carboxylic-groups at the o/w interface, which cause a higher interfacial density of naphthenic acid monomers. The permanent lowering in IFT is likely due to formation of positively charged monoacid complexes, which possess high interfacial activity. On the other hand, in the case of the aromatic model compounds, the cations affected the IFT differently. This is mainly discussed in light of degree of cation hydration and steric conditions. Various oil-soluble non-ionic surfactant mixtures were also introduced to systems involving a model naphthenic acid and Ca²⁺ in order to investigate how the interfacial competition affected the local interactions. Based on the behaviour of dynamic IFT, probable inhibition mechanisms are discussed.Electronic Supplementary Material Supplementary material is available for this article at