Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Introduction of a fluorine moiety into green fluorescent protein offers an interesting novel spectral variant. The calculated binding energy of fluorotyrosine (F-Tyr) (−8.42 kcal/mol) for tyrosyl tRNA synthetase was moderately higher than that of tyrosine (Tyr) (−8.36 kcal/mol). This result directly correlated with the expression level of F-Tyr containing GFP (38 mg/l), which was comparably higher than that of the parent GFP expression level (34 mg/l). Finally, we generated a model structure for GFP to assess possible interaction in the chromophore of the protein structure, which plays an important role in determining the spectral and folding behaviors of the F-Tyr incorporated GFP variant.     

The distribution of dimethylsulfoniopropionate in tropical Pacific coral reef invertebrates

Dimethylsulfoniopropionate (DMSP) is an important component of the global sulfur cycle and may be involved, via its cleavage product dimethylsulfide, in climate regulation. Although it is common in many algae, reports of DMSP in animals, particularly tropical invertebrates, are limited. This study examined the distribution of DMSP in a diverse group of coral reef invertebrates. DMSP was present in all 22 species of cnidarians and ranged from 9 to 723 μmol g⁻¹ of dry mass (DM) with a mean (± 1SD) of 110 ± 180 μmol g⁻¹ DM. It was not detected in a flatworm and an ascidian or in two of five sponges. Concentrations in sponges ranged from undetectable to 16 μmol g⁻¹ DM with a mean of 4 ± 7 μmol g⁻¹ DM. Within the cnidarians, DMSP concentrations did not differ among orders. Among cnidarian species, DMSP concentrations were correlated with symbiotic zooxanthellae densities. Within cnidarian species, DMSP concentrations of individuals were positively correlated with zooxanthellae densities in three of the four species examined. We speculate that DMSP is dietarily derived in sponges and derived from zooxanthellae in the cnidarians. The functions of DMSP in coral reef invertebrates are not known.     

The internal rotational barriers about NCα and CαC backbone bonds of polypeptides

In many studies on the protein folding problem it is assumed that the internal rotational barriers about NC^α and C^αC backbone bonds in unfolded polypeptides are quite small, around 0.7 kcal/mol, of an order comparable to the energy of kT at normal temperature (where k is Boltzmann’s constant and T is the temperature in K) and hence that rotations about these bonds occur almost freely. Here it is highlighted that such consideration is an unfortunate mistake. Approximate values for the rotational barriers of NC^α and C^αC bonds are suggested from computations of U(f \phi , ψ) potential energy surface (PES) maps of a number of oligopeptides by a semiempirical method for conformational analysis. The proposed values are about 16 kcal/mol for NC^α bonds and 6 kcal/mol for C^αC bonds. The values of the same barriers estimated from some ab initio quantum-mechanical PES maps for several dipeptides available in literature are also highlighted.     

Engineering heme binding sites in monomeric rop

Heme ligands were introduced in the hydrophobic core of an engineered monomeric ColE1 repressor of primer (rop-S55) in two different layers of the heptad repeat. Mutants rop-L63M/F121H (layer 1) and rop-L56H/L113H (layer 3) were found to bind heme with a K _D of 1.1 ± 0.2 and 0.47 ± 0.07 μM, respectively. The unfolding of heme-bound and heme-free mutants, in the presence of guanidinium hydrochloride, was monitored by both circular dichroism and fluorescence spectroscopy. For the heme-bound rop mutants, the total free energy change was 0.5 kcal/mol higher in the layer 3 mutant compared with that in the layer1 mutant. Heme binding also stabilized these mutants by increasing the by 1.4 and 1.8 kcal/mol in rop-L63M/F121H and rop-L56H/L113H, respectively. The reduction potentials measured by spectroelectrochemical titrations were calculated to be −154 ± 2 mV for rop-56H/113H and −87.5 ± 1.2 mV for rop-L63M/F121H. The mutant designed to bind heme in a more buried environment (layer 3) showed tighter heme binding, a higher stability, and a different reduction potential compared with the mutant designed to bind heme in layer 1.     

Conversion of oleic acid to 10-hydroxystearic acid by whole cells of <Emphasis Type="Italic">Stenotrophomonas nitritireducens</Emphasis>

The optimal reaction conditions for the conversion of oleic acid to 10-hydroxystearic acid by whole cells of Stenotrophomonas nitritireducens were: pH 7.5, 35°C, 0.05% (w/v) Tween 80, 20 g cells l⁻¹, and 30 g oleic acid l⁻¹ in an anaerobic atmosphere. Under these conditions, the cells produced 31.5 g 10-hydroxystearic acid l⁻¹ over 4 h with a conversion yield of 100% (mol/mol) and a productivity of 7.9 g l⁻¹ h⁻¹, indicating that oleic acid was converted completely to 10-hydroxystearic acid, with no detectable byproduct. This is the highest concentration, productivity, and yield of 10-hydroxystearic acid from oleic acid reported thus far.     

Understanding structural/functional properties of amidase from Rhodococcus erythropolis by computational approaches

The 3D structure of the amidase from Rhodococcus erythropolis (EC 3.5.1.4) built by homology-based modeling is presented. Propionamide and acetamide are docked to the amidase. The reaction models were used to characterize the explicit enzymatic reaction. The calculated free energy barrier at B3LYP/6-31G* level of Model A (Ser194 + propionamide) is 19.72 kcal mol⁻¹ in gas (6.47 kcal mol⁻¹ in solution), and of Model B (Ser194 + Gly193 + propionamide) is 18.71 kcal mol⁻¹ in gas (4.57 kcal mol⁻¹ in solution). The docking results reveal that propionamide binds more strongly than acetamide due to the ethyl moiety of propionamide, which makes the carboxyl oxygen center of the substrate slightly more negative, making formation of the positively charged tetrahedral intermediate slightly easier. The quantum mechanics results demonstrate that Ser194 is essential for the acyl-intermediate, and Gly193 plays a secondary role in stabilizing acyl-intermediate formation as the NH groups of Ser194 and Gly193 form hydrogen bonds with the carbonyl oxygen of propionamide. The new structural and mechanistic insights gained from this computational study should be useful in elucidating the detailed structures and mechanisms of amidase and other homologous members of the amidase signature family.     

Initial step of B12-dependent enzymatic catalysis: energetic implications regarding involvement of the one-electron-reduced form of adenosylcobalamin cofactor

Density functional theory analysis was performed to elucidate the impact of one-electron reduction upon the initial step of adenosylcobalamin-dependent enzymatic catalysis. The transition state (TS) corresponding to the Co–C bond cleavage and subsequent hydrogen abstraction from the substrate was located. The intrinsic reaction coordinate calculations predicted that the reaction consisting of Co–C5′ bond cleavage in [Co^III(corrin^•)]–Rib (where Rib is ribosyl) and hydrogen-atom abstraction from the CH₃–CH₂–CHO substrate occurs in a concerted fashion. The computed activation energy barrier of the reaction (15.0 kcal/mol) was lowered by approximately 54.5% in comparison with the reaction involving the positively charged cofactor model (Im–[Co^III(corrin)]–Rib⁺, where Im is imidazole; energy barrier = 33.0 kcal/mol). The Im base was detached during the TS search in the reaction involving the one-electron-reduced analogue. Thus, to compare the energetics of the two reactions, the axial Im ligand detachment energy for the Im–[Co^III(corrin^•)]–Rib model was computed [7.6 kcal/mol (gas phase); 4.6 kcal/mol (water)]. Consequently, the effective activation energy barrier for the reaction mediated by the Im-off [Co^III(corrin^•)]–Rib was estimated to be 22.6 kcal/mol, which implied an overall 31.5% reduction in the energetic demands of the reaction. Considering that the lengthened Co–N_axial bond has been observed in X-ray crystal structure studies of B₁₂-dependent mutases, the catalytic impact induced by one-electron reduction of the cofactor is expected to be higher in the presence of the enzymatic environment.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Fermentation of high concentrations of lactose to ethanol by engineered flocculent Saccharomyces cerevisiae

The development of microorganims that efficiently ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. The lactose fermentation performance of a recombinant Saccharomyces cerevisiae flocculent strain was evaluated. The yeast consumed rapidly and completely lactose concentrations up to 150 g l⁻¹ in either well- or micro-aerated batch fermentations. The maximum ethanol titre was 8% (v/v) and the highest ethanol productivity was 1.5–2 g l⁻¹ h⁻¹, in micro-aerated fermentations. The results presented here emphasise that this strain is an interesting alternative for the production of ethanol from lactose-based feedstocks.     

Fluvastatin inhibits angiotensin II-induced nuclear factor kappa B activation in renal tubular epithelial cells through the p38 MAPK pathway

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been shown to reduce the progression of renal disease independent of cholesterol-lowering effect, but the mechanism of potential protective effect remains unclear. Here, we investigate the effect of fluvastatin on activation of nuclear factor-κB (NF-κB) induced by angiotensin II (AngII) in rat kidney tubule epithelial cells (NRK-52E). Electrophoretic mobility shift assays (EMSA) was used to detect NF-κB activation. Phosphorylation of cellular p38 mitogen-activated protein kinase (p38MAPK) was determined by western blot analysis. AngII stimulated the DNA-binding activity of NF-κB and phosphorylation of p38MAPK in cultured NRK-52E cells in a dose-dependent (10⁻⁹–10⁻⁶ mol/l) manner (P < 0.01). AngII (10⁻⁶ mol/l) induced a rapid (5 min) increase of the p38MAPK phosphorylation. NF-κB DNA-binding activity was increased at as early as 30 min, peaked at 2 h after AngII treatment. This stimulatory effect of AngII on NF-κB was blocked by SB203580 (a specific inhibitor of p38MAPK). Incubation of cells with fluvastatin significantly inhibited the AngII-induced NF-κB activation in a dose-dependent (10⁻⁷–10⁻⁵ mol/l) manner (P < 0.05). Exogenous mevalonate (10⁻⁴mol/l) prevented the effect of fluvastatin on NF-κB activation. These results suggest the fluvastatin reduced AngII-induced NF-κB activation via the p38MAPK pathway in NRK-52E cells. The effect is at least partly due to blocking the biosynthesis of mevalonate.     

Micropropagation of <Emphasis Type="Italic">Pinus massoniana</Emphasis> and mycorrhiza formation in vitro

A protocol was developed for the micropropagation of Pinus massoniana and mycorrhiza formation on rooted microshoots. Seedling explants were first cultured on Gresshoff and Doy (GD) medium supplemented with 6-benzyladenine (BA) alone or in combination with α-napthaleneacetic acid (NAA) to stimulate the formation of intercotyledonary axillary buds. The frequency of axillary bud induction was up to 97% on medium supplemented with 4.0 mg l⁻¹ BA and 0. 2 mg l⁻¹ NAA, and the average number of buds per explant reached up to 5.5 on medium with 4.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Axillary buds elongated rapidly after being transferred to half-strength GD medium containing activated charcoal (0.1% w/v). Shoot proliferation was achieved by cutting elongated shoots into stem segments and subculturing on GD medium containing 2 mg l⁻¹ BA and 0.2 mg l⁻¹ NAA. Root primordia were induced in 82% of shoots when transferred to half-strength GD medium containing 0.2 mg l⁻¹ NAA. Root elongation was achieved in a hormone-free GD agar medium or a perlite substrate. Rooted plantlets were inoculated with the mycelium of ectomycorrhizal fungus Pisolithus tinctorius and the formation of ectomycorrhiza-like structures was achieved in vitro.     

Macrophyte beds contribute disproportionately to benthic invertebrate abundance and biomass in a sand plains stream

Macrophyte beds have been shown to influence organic matter retention and nutrient processing in streams. Less is known about the extent to which plant beds contribute to abundance, biomass, and diversity of macroinvertebrate assemblages in low-order streams. We measured aquatic invertebrate abundance, biomass, and diversity associated with plant beds and sand/gravel patches in a low-gradient second-order stream in the Central Sand Plains of Wisconsin, USA from March to October. Invertebrate abundance and biomass were higher on average in plant beds (2,552 m⁻² and 1,575 mg m⁻²) than in sand/gravel patches (893 m⁻² and 486 mg m⁻²). Although sand/gravel habitat was over three times more abundant than plant beds in the study reach, plant beds and sand/gravel patches contributed similarly to invertebrate abundance and biomass at the whole-reach scale. The abundance and biomass of invertebrates associated with plant beds decreased from spring to autumn. Non-insect invertebrates in the plant beds increased in relative abundance as the year progressed. Shannon–Weiner diversity and taxa richness of invertebrates were higher in the plant beds than in the sand/gravel habitat. Our results suggest that plant beds can represent hot spots for invertebrate abundance and production in low-gradient streams, and have implications for stream management and restoration in these types of ecosystems. Handling editor: S. I. Dodson     

Theoretical investigation of tautomerism in N-hydroxy amidines

A DFT study with QST3 approach method is used to calculate kinetic, thermodynamic, spectral and structural data of tautomers and transition state structures of some N-hydroxy amidines. All tautomers and transition states are optimized at the B3LYP/6-311++g** and B3LYP/aug-cc-pvtz level, with good agreement in energetic result with energies obtained from CBS-QB3, a complete basis set composite energy method. The result shows that the tautomer a (amide oxime) is more stable than the tautomer b (imino hydroxylamine) as is reported in the literature. In addition, our finding shows that, the energy difference between two tautomers is only in about 4–10 kcal/mol but the barrier energy found in traversing each tautomer to another one is in the range of 33–71 kcal/mol. Therefore, it is impossible to convert these two tautomers to each other at room temperature. Additionally, transition state theory is applied to estimate the barrier energy and reaction rate constants of the hydrogen exchange between tautomers in presence of 1–3 molecules of water. The computed activation barrier shows us that the barrier energy of solvent assisted tautomerism is about 9–20 kcal/mol and lower than simple tautomerism and this water-assisted tautomerism is much faster than simple tautomerism, especially with the assisting two molecules of water.     

Stimulation of reductive glycerol metabolism by overexpression of an aldehyde dehydrogenase in a recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strain defective in the oxidative pathway

Previously, we constructed a glycerol oxidative pathway-deficient mutant strain of Klebsiella pneumoniae by inactivation of glycerol dehydrogenase (dhaD) to eliminate by-product synthesis during production of 1,3-propanediol (1,3-PD) from glycerol. Although by-product formation was successfully blocked in the resultant strain, the yield of 1,3-PD was not enhanced, probably because dhaD disruption resulted in insufficient regeneration of the cofactor NADH essential for the activity of 1,3-PD oxidoreductase (DhaT). To improve cofactor regeneration, in the present study we overexpressed an NAD⁺-dependent aldehyde dehydrogenase in the recombinant strain. To this end, an aldehyde dehydrogenase AldHk homologous to E. coli AldH but with NAD⁺-dependent propionaldehyde dehydrogenase activity was identified in K. pneumoniae. Functional analysis revealed that the substrate specificity of AldHk embraced various aldehydes including propionaldehyde, and that NAD⁺ was preferred over NADP⁺ as a cofactor. Overexpression of AldHk in the glycerol oxidative pathway-deficient mutant AK/pVOTHk resulted in a 3.6-fold increase (0.57 g l⁻¹ to 2.07 g l⁻¹) in the production of 3-hydroxypropionic acid (3-HP), and a 1.1-fold enhancement (8.43 g l⁻¹ to 9.65 g l⁻¹) of 1,3-PD synthesis, when glycerol was provided as the carbon source, compared to the levels synthesized by the control strain (AK/pVOT). Batch fermentation using AK/pVOTHk showed a significant increase (to 70%, w/w) in conversion of glycerol to the reductive metabolites, 1,3-PD and 3-HP, with no production of by-products except acetate.     

Mechanism of peptide hydrolysis by co-catalytic metal centers containing leucine aminopeptidase enzyme: a DFT approach

In this density functional theory study, reaction mechanisms of a co-catalytic binuclear metal center (Zn1–Zn2) containing enzyme leucine aminopeptidase for two different metal bridging nucleophiles (H₂O and –OH) have been investigated. In addition, the effects of the substrate (l-leucine-p-nitroanilide → l-leucyl-p-anisidine) and metal (Zn1 → Mg and Zn2 → Co, i.e., Mg1–Zn2 and Mg1–Co2 variants) substitutions on the energetics of the mechanism have been investigated. The general acid/base mechanism utilizing a bicarbonate ion followed by this enzyme is divided into two steps: (1) the formation of the gem-diolate intermediate, and (2) the cleavage of the peptide bond. With the computed barrier of 17.8 kcal/mol, the mechanism utilizing a hydroxyl nucleophile was found to be in excellent agreement with the experimentally measured barrier of 18.7 kcal/mol. The rate-limiting step for reaction with l-leucine-p-nitroanilide is the cleavage of the peptide bond with a barrier of 17.8 kcal/mol. However, for l-leucyl-p-anisidine all steps of the mechanism were found to occur with similar barriers (18.0–19.0 kcal/mol). For the metallovariants, cleavage of the peptide bond occurs in the rate-limiting step with barriers of 17.8, 18.0, and 24.2 kcal/mol for the Zn1–Zn2, Mg1–Zn2, and Mg1–Co2 enzymes, respectively. The nature of the metal ion was found to affect only the creation of the gem-diolate intermediate, and after that all three enzymes follow essentially the same energetics. The results reported in this study have elucidated specific roles of both metal centers, the nucleophile, indirect ligands, and substrates in the catalytic functioning of this important class of binuclear metallopeptidases.     

Kinetics of CO conversion into H<Subscript>2</Subscript> by <Emphasis Type="Italic">Carboxydothermus hydrogenoformans</Emphasis>

The objective of this study was to improve the biological water–gas shift reaction for producing hydrogen (H₂) by conversion of carbon monoxide (CO) using an anaerobic thermophilic pure strain, Carboxydothermus hydrogenoformans. Specific hydrogen production rates and yields were investigated at initial biomass densities varying from 5 to 20 mg volatile suspended solid (VSS) L⁻¹. Results showed that the gas–liquid mass transfer limits the CO conversion rate at high biomass concentrations. At 100-rpm agitation and at CO partial pressure of 1 atm, the optimal substrate/biomass ratio must exceed 5 mol CO g⁻¹ biomass VSS in order to avoid gas–liquid substrate transfer limitation. An average H₂ yield of 94 ± 3% and a specific hydrogen production rate of ca. 3 mol g⁻¹ VSS day⁻¹ were obtained at initial biomass densities between 5 and 8 mg VSS⁻¹. In addition, CO bioconversion kinetics was assessed at CO partial pressure from 0.16 to 2 atm, corresponding to a dissolved CO concentration at 70°C from 0.09 to 1.1 mM. Specific bioactivity was maximal at 3.5 mol CO g⁻¹ VSS day⁻¹ for a dissolved CO concentration of 0.55 mM in the culture. This optimal concentration is higher than with most other hydrogenogenic carboxydotrophic species.     

Micropropagation of <Emphasis Type="Italic">Cotoneaster wilsonii</Emphasis> Nakai—a rare endemic ornamental plant

A simple and efficient micropropagation system was developed for Cotoneaster wilsonii through node and shoot tip explants obtained from mature field-grown plants. Of the two explants, node explants were found to be the most effective for axillary shoot proliferation. The highest frequency of shoot induction was achieved when nodal explants were incubated on Murashige and Skoog (MS) medium supplemented with 0.5 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ α- naphthaleneacetic acid (NAA) with an average of 34 shoots per explant. The microshoots were separated from the multiple shoots and subcultured on MS medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar for further shoot growth. Maximum rooting was obtained on half-strength MS medium supplemented with 0.5 mg L⁻¹ indole-3-butyric acid (IBA). The in vitro-grown plantlets were successfully acclimatized in a glasshouse with 98% of survival. High concentrations of TDZ (1.5–2.0 mg L⁻¹) and repeated subcultures resulted hyperhydric shoots. Supplementation of the culture medium with silicon significantly reduced the induction of hyperhydric shoots. Increasing silicon concentration significantly decreased malondialdehyde content of the regenerated shoots. Data indicate that addition of silicon to the culture medium can effectively control hyperhydricity.     

Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·10⁶ M⁻¹≤K≤1.4·10⁶ M⁻¹, free energy ΔG ^o=−8.7±0.3 kcal/mol, enthalpy ΔH ^o≅0, and entropy ΔS ^o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T _m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10⁻⁵ M EtBr) increased by 17°C as compared with the ΔT _m of free homopolymer, whereas the half-width of the transition (T _m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K ₁=1.7·10⁵ M⁻¹; ΔG ^o=−8.10±0.03 kcal/mol) and weak (K ₂=2.9·10³ M⁻¹; ΔG ^o=−6.0±0.3 kcal/mol).The ΔG ^o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.     

The molecular basis for the inhibition of human cytochrome P450 1A2 by oroxylin and wogonin

In our previous kinetics studies the natural products oroxylin and wogonin were shown to have strong biological affinity for, and inhibitory effects against, human cytochrome P450 1A2, with IC₅₀ values of 579 and 248 nM, respectively; this might lead to the occurrence of drug–drug interactions when co-administered clinically. However, their inhibitory mechanisms against 1A2 remain elusive. In this study, molecular docking and molecular dynamics simulations were performed to better understand the molecular basis of their inhibitory mechanisms towards 1A2. Structural analysis revealed that oroxylin has a different binding pattern from wogonin and another very strongly binding inhibitor α-naphthoflavone (ANF, IC₅₀ = 49 nM). The O₇ atom of oroxylin forms hydrogen bonds with the OD1/OD2 atoms of Asp313, which is not observed in the 1A2–wogonin complex. Because of energetically unfavorable repulsions with the methoxy group at the 6 position of the oroxylin ring, significant conformational changes were observed for the sidechain of Thr118 in the MD simulated model. As a result, the larger and much more open binding-site architecture of the 1A2–oroxylin complex may account for its weaker inhibitory effect relative to the 1A2–ANF complex. Energy analysis indicated that oroxylin has a less negative predicted binding free energy of −19.8 kcal/mol than wogonin (−21.1 kcal/mol), which is consistent with our experimental assays. Additionally, our energy results suggest that van der Waals/hydrophobic and hydrogen-bonding interactions are important in the inhibitory mechanisms of oroxylin whereas the former is the underlying force responsible for strong inhibition by ANF and wogonin.     

Kinetic and thermodynamic analysis of dimerization inhibitors binding to HIV protease monomers by surface plasmon resonance

The analysis of kinetic and thermodynamic parameters of binding of peptide and nonpeptide dimerization inhibitors of HIV protease (HIVp) to the enzyme monomers immobilized on an optical chip has been studied by surface plasmon resonance. The molecular interactions were investigated at different inhibitor concentrations (0–80 μM) and temperatures (15–35°C). Determination of kinetic (k _on, k _off), equilibrium (K _d), and thermodynamic (ΔG, ΔH, and -TΔS) has shown that both inhibitors are characterized by similar interaction parameters and the entropic term (-TΔS) of about −20 kcal/mol is the main driving force for the HIVp complex formation with the inhibitors, while the positive value (14 kcal/mol) of the enthalpic term (ΔH) counteracted the complex formation.