Effect of the Addition of Soybean Residue (Okara) on the Physicochemical, Tribological, Instrumental, and Sensory Texture Properties of Extruded Snacks.

An extrusion process was used to improve the physical and textural characteristics of an extruded snack supplemented with soybean residue (okara). An extreme vertices mixture design with a constraint for okara flour (0−50%), mung bean flour (20−70%), and rice flour (20−80%) resulted in the production of eleven formulations. The color, radial expansion index (REI), bulk density, tribological behavior, and instrumental and sensory texture of the extruded snacks were evaluated. Increasing the quantity of okara resulted in an extrudate with a darker, redder color, decreased REI, increased bulk density, and decreased crispness. The tribological pattern of the snack was determined by its dominant composition (protein, starch, or fiber) in the flour mixture, which contributed to the stability of the lubricating film under rotational shear. A principal component analysis of sensory data captured a total of 81.9% variations in the first two dimensions. Texture appeal was inversely related to tooth packing (r = −0.646, p < 0.05). The optimized formulation for texture preference had an okara content of 19%, which was 104% crispier and 168% tougher than an okara content of 40%. This by-product of soybean milk processing can thus be used to develop gluten-free snacks with desirable physical characteristics and texture.

Simulation of oleuropein structural conformation in vacuum, water and triolein-water systems using molecular dynamics.

Oleuropein, the main phenolic compound of olive leaves, exhibits a unique blend of biological activities and has been shown to locate itself at the oil-water (O/W) interface. This behavior could influence the physico-chemical properties of dispersed systems such as emulsions. In this work, we study the effect of the microenvironment (vacuum, water, and triolein-water) on the conformational preferences of oleuropein using molecular dynamics (MD) simulations at 300K for at least 30ns. The seven torsions that describe the flexible skeleton of oleuropein were monitored together with the distance between the glucose (Glu) and hydroxytyrosol (Hyd) moieties (dglu-hyd) of the molecule. The obtained trajectories demonstrated that oleuropein adopts different conformations that depend on the environment. The preferential conformers in each system were analyzed for their molecular geometry and internal energy. In vacuum, the oleuropein preferential conformation is tight with the glucose moiety in close proximity with the hydroxytyrosol moiety. In water, oleuropein preferential conformers presented large differences in their structural properties, varying from a close like U form, and a semi-opened form, to an opened form characterized by high fluctuations in dglu-hyd values. In a triolein-water system, oleuropein tends to adopt a more open form where the glucose moiety could be approximately aligned with the hydroxytyrosol and elenolic acid moieties. Based on a calculation at the HF/6-31G* level, these flexibilities of oleuropein required energy of 19.14kcal/mol in order to adopt the conformation between water and triolein-water system. A radial distribution function (RDF) analysis showed that specific hydroxyl groups of Hyd and Glu interact with water molecules, enabling us to understand the amphiphilic character of oleuropein at the triolein-water interface. MD calculations together with interfacial tension measurements revealed that the oleuropein binding at O/W interface is an enthalpy driven mechanism.

Elucidation of hydroxyl groups-antioxidant relationship in mono- and dihydroxyflavones based on O-H bond dissociation enthalpies.

Radical scavenging potential is the key to anti-oxidation of hydroxyflavones which generally found in fruits and vegetables. The objective of this work was to investigate the influence of hydroxyl group on the O-H bond dissociation enthalpies (BDE) from a series of mono- and dihydroxyflavones. Calculation at the B3LYP/6-31G(d,p) level reveals the important roles of an additional one hydroxyl group to boost the BDE of hydroxyflavones that were a stabilization of the generated radicals through attractive H-bond interactions, an ortho- and para-dihydroxyl effect, and a presence of the 3-OH in dihydroxyflavones. On the other hand, the meta-dihydroxyl effect and range-hydroxyl effect especially associated with the either 5-OH or 8-OH promoted greater BDE. Results did not only confirm that dihydroxyflavones had lower BDE than monohydroxyflavones but also suggest the selective potent hydroxyflavone molecules that are the 6'-hydroxyflavone (for monohydroxyflavone) and the 5',6'-, 7,8- and 3',4'-dihydroxyflavone which the corresponding radical preferable generated at C6'-O•, C8-O• and C4'-O•, respectively. Electron distribution was limited only over the two connected rings of hydroxyflavones while the expansion distribution into C-ring could be enhanced if the radical was formed especially for the 2',3'- and 5',6'dihydroxyflavone radicals. The delocalized bonds were strengthened after radical was generated. However the 5-O• in 5,6-dihydroxyflavone and the 3-O• in 3,6'-dihydroxyflavone increased the bond order at C4-O11 which might interrupt the conjugated delocalized bonds at the keto group.

Mechanistic insights into the catalytic reaction of plant allene oxide synthase (pAOS) via QM and QM/MM calculations.

QM cluster and QM/MM protein models have been employed to understand aspects of the reaction mechanism of plant allene oxide synthase (pAOS). In this study we have investigated two reaction mechanisms for pAOS. The standard pAOS mechanism was contrasted with an alternative involving an additional active site molecule which has been shown to facilitate proton coupled electron transfer (PCET) in related systems. Firstly, we found that the results from QM/MM protein model are comparable with those from the QM cluster model, presumably due to the large active site used. Furthermore, the results from the QM cluster model show that the Fe(III) and Fe(IV) pathways for the standard mechanism have similar energetic and structural properties, indicating that the reaction mechanism may well proceed via both pathways. However, while the PCET process is facilitated by an additional active site bound water in other related families, in pAOS it is not, suggesting this type of process is not general to all closely related family members.

Dissecting substrate specificity of two rice BADH isoforms: Enzyme kinetics, docking and molecular dynamics simulation studies.

Fragrance rice (Oryza sativa) contains two isoforms of BADH, named OsBADH1 and OsBADH2. OsBADH1 is implicated in acetaldehyde oxidation in rice plant peroxisomes, while the non-functional OsBADH2 is believed to be involved in the accumulation of 2-acetyl-1-pyrroline, the major compound of aroma in fragrance rice. In the present study, site-directed mutagenesis, molecular docking and molecular dynamics simulation studies were used to investigate the substrate specificity towards Bet-ald and GAB-ald. Consistent with our previous study, kinetics data indicated that the enzymes catalyze the oxidation of GAB-ald more efficiently than Bet-ald and the OsBADH1 W172F and OsBADH2 W170F mutants displayed a higher catalytic efficiency towards GAB-ald. Molecular docking analysis and molecular dynamics simulations for the first time provided models for aldehyde substrate-bound complexes of OsBADHs. The amino acid residues, E262, L263, C296 and W461 of OsBADH1 and E260, L261, C294 and W459 of OsBADH2 located within 5 Å of the OsBADH active site mainly interacted with GAB-ald forming strong hydrogen bonds in both OsBADH isoforms. Residues W163, N164, Q294, C296 and F397 of OsBADH1-Bet-ald and Y163, M167, W170, E260, S295 and C453 of OsBADH2-Bet-ald formed the main interaction sites while E260 showed an interaction energy of -14.21 kcal/mol. Unconserved A290 in OsBADH1 and W288 in OsBADH2 appeared to be important for substrate recognition similar to that observed in PsAMADHs. Overall, the results here help to explain how two homologous rice BADHs recognize the aldehyde substrate differently, a key property to their biological role.

Characteristic vibration patterns of odor compounds from bread-baking volatiles upon protein binding: density functional and ONIOM study and principal component analysis.

As the mechanism underlying the sense of smell is unclear, different models have been used to rationalize structure-odor relationships. To gain insight into odorant molecules from bread baking, binding energies and vibration spectra in the gas phase and in the protein environment [7-transmembrane helices (7TMHs) of rhodopsin] were calculated using density functional theory [B3LYP/6-311++G(d,p)] and ONIOM [B3LYP/6-311++G(d,p):PM3] methods. It was found that acetaldehyde ("acid" category) binds strongly in the large cavity inside the receptor, whereas 2-ethyl-3-methylpyrazine ("roasted") binds weakly. Lys296, Tyr268, Thr118 and Ala117 were identified as key residues in the binding site. More emphasis was placed on how vibrational frequencies are shifted and intensities modified in the receptor protein environment. Principal component analysis (PCA) suggested that the frequency shifts of C-C stretching, CH(3) umbrella, C = O stretching and CH(3) stretching modes have a significant effect on odor quality. In fact, the frequency shifts of the C-C stretching and C = O stretching modes, as well as CH(3) umbrella and CH(3) symmetric stretching modes, exhibit different behaviors in the PCA loadings plot. A large frequency shift in the CH(3) symmetric stretching mode is associated with the sweet-roasted odor category and separates this from the acid odor category. A large frequency shift of the C-C stretching mode describes the roasted and oily-popcorn odor categories, and separates these from the buttery and acid odor categories.

In vitro and in silico binding study of the peptide derived from HIV-1 CA-CTD and LysRS as a potential HIV-1 blocking site.

The assembly process in HIV-1 has become a new target for infected HIV-1 patient treatment. During this process, the viral genomic RNA and precursor protein are assembled at the permeable membrane and tRNA(Lys3) is packed into a new virion as the primer for the reverse transcription process. The packaging of tRNA(Lys3) arises from the interaction of HIV-1 Gag and hLysRS. To better understand the formation of this ternary complex, the interaction study of LysRS-peptide complex using a combination of circular dichroism, molecular dockings and molecular dynamic simulations are reported here. The circular dichroism experiments confirm that the sh-H4 peptide, containing 10 amino acid residues from helix4 of C-terminal domain of HIV-1 capsid protein (CA-CTD), can be induced to form a helical structure upon binding to hLysRS. Molecular docking analysis of LysRS (hLysRS and eLysRS) with the sh-H4 peptide revealed the two possible arrangements of the peptide upon the binding event. Molecular dynamics based free energy calculations of the peptide binding process are used to determine the interactions as well as the important amino acid residues involving in binding. The peptide is found to lie against helix 7 of LysRS in a perpendicular fashion. Additionally, the peptide preferably interacts with hLysRS over eLysRS including strong hydrogen bond interactions between R247-Q219 and R241-E212. Interestingly, these amino acid residues are found in both LysRS and CA-CTD. These important residues appear to be a vital feature of the LysRS-CA-CTD complex and may ultimately lead to the inhibitor design to block the Gag-LysRS interaction.

Ranking ligand affinity for the DNA minor groove by experiment and simulation.

The structural and thermodynamic basis for the strength and selectivity of the interactions of minor groove binders (MGBs) with DNA is not fully understood. In 2003, we reported the first example of a thiazole-containing MGB that bound in a phase-shifted pattern that spanned six base pairs rather than the usual four (for tricyclic distamycin-like compounds). Since then, using DNA footprinting, NMR spectroscopy, isothermal titration calorimetry, and molecular dynamics, we have established that the flanking bases around the central four being read by the ligand have subtle effects on recognition. We have investigated the effect of these flanking sequences on binding and the reasons for the differences and established a computational method to rank ligand affinity against varying DNA sequences.

Bridge water mediates nevirapine binding to wild type and Y181C HIV-1 reverse transcriptase--evidence from molecular dynamics simulations and MM-PBSA calculations.

The important role of the bridge water molecule in the binding of HIV-1 reverse transcriptase (RT) inhibitor complex was elucidated by molecular dynamics (MD) simulations using an MM-PBSA approach. Binding free energies and thermodynamic property differences for nevirapine bound to wild type and Y181C HIV-1 reverse transcriptase were investigated, and the results were compared with available experimental data. MD simulations over 3 ns revealed that the bridge water formed three characteristic hydrogen bonds to nevirapine and two residues, His235 and Leu234, in the binding pocket. The energetic derived model, which was determined from the consecutive addition of a water molecule, confirmed that only the contribution from the bridge water was essential in the binding configuration. Including this bridge water in the MM-PBSA calculations reoriented the binding energies from -32.20 to -37.65 kcal/mol and -28.07 to -29.82 kcal/mol in the wild type and Y181C HIV-1 RT, respectively. From the attractive interactions via the bridge water, His235 and Leu234 became major contributions. We found that the bridge water is the key in stabilizing the bound complex; however, in the Y181C RT complex this bridge water showed weaker hydrogen bond formation, lack of attractive force to nevirapine and lack of binding efficiency, leading to the failure of nevirapine against the Y181C HIV-1 RT. Moreover, the dynamics of Val179, Tyr181Cys, Gly190 and Leu234 in the binding pocket showed additional attractive energetic contributions in helping nevirapine binding. These findings that the presence of a water molecule in the hydrophobic binding site plays an important role are a step towards a quantitative understanding of the character of bridge water in enzyme-inhibitor binding. This can be helpful in developing designs for novel non-nucleoside HIV-1 RT inhibitors active against the mutant enzyme.

A detailed binding free energy study of 2:1 ligand-DNA complex formation by experiment and simulation.

In 2004, we used NMR to solve the structure of the minor groove binder thiazotropsin A bound in a 2:1 complex to the DNA duplex, d(CGACTAGTCG)2. In this current work, we have combined theory and experiment to confirm the binding thermodynamics of this system. Molecular dynamics simulations that use polarizable or non-polarizable force fields with single and separate trajectory approaches have been used to explore complexation at the molecular level. We have shown that the binding process invokes large conformational changes in both the receptor and ligand, which is reflected by large adaptation energies. This is compensated for by the net binding free energy, which is enthalpy driven and entropically opposed. Such a conformational change upon binding directly impacts on how the process must be simulated in order to yield accurate results. Our MM-PBSA binding calculations from snapshots obtained from MD simulations of the polarizable force field using separate trajectories yield an absolute binding free energy (-15.4 kcal mol(-1)) very close to that determined by isothermal titration calorimetry (-10.2 kcal mol(-1)). Analysis of the major energy components reveals that favorable non-bonded van der Waals and electrostatic interactions contribute predominantly to the enthalpy term, whilst the unfavorable entropy appears to be driven by stabilization of the complex and the associated loss of conformational freedom. Our results have led to a deeper understanding of the nature of side-by-side minor groove ligand binding, which has significant implications for structure-based ligand development.