Biomolecular interaction of purified recombinant Arabidopsis thaliana's alternative oxidase 1A with TCA cycle metabolites: Biophysical and molecular docking studies.

In higher plants, the mitochondrial alternative oxidase (AOX) pathway plays an essential role in maintaining the TCA cycle/cellular carbon and energy balance under various physiological and stress conditions. Though the activation of AOX pathway upon exogenous addition of α-ketoacids/TCA cycle metabolites [pyruvate, α-ketoglutarate (α-KG), oxaloacetic acid (OAA), succinate and malic acid] to isolated mitochondria is known, the molecular mechanism of interaction of these metabolites with AOX protein is limited. The present study is designed to understand the biomolecular interaction of pure recombinant Arabidopsis thaliana AOX1A with TCA cycle metabolites under in vitro conditions using various biophysical and molecular docking studies. The binding of α-KG, fumaric acid and OAA to rAtAOX1A caused conformational change in the microenvironment of tryptophan residues as evidenced by red shift in the synchronous fluorescence spectra (∆λ = 60 nm). Besides, a decrease in conventional fluorescence emission spectra, tyrosine specific synchronous fluorescence spectra (∆λ = 15 nm) and α-helical content of CD spectra revealed the conformation changes in rAtAOX1A structure associated with binding of various TCA cycle metabolites. Further, surface plasmon resonance (SPR) and microscale thermophoresis (MST) studies revealed the binding affinity, while docking studies identified binding pocket residues, respectively, for these metabolites on rAtAOX1A.

Immobilization of Cellulase Enzymes on Single-Walled Carbon Nanotubes for Recycling of Enzymes and Better Yield of Bioethanol Using Computer Simulations.

The utilization of microbial cellulase enzymes for transforming plant biomass into biofuel or bioethanol, which can serve as a substitute for fossil fuel, is a subject of growing interest. Nonetheless, large-scale production of biofuel using cellulases is not economically feasible as the extraction of these enzymes from diverse microorganisms is an expensive process. To address this issue, immobilizing the enzyme to a substrate material, e.g., carbon nanotubes (CNTs), to recycle without a significant decline in its catalytic activity is a promising solution. Due to the hydrophobic nature of CNTs, we employed molecular docking and network analysis methodologies to identify potential CNT-binding sites on the outer surface of a wild-type cellulase enzyme, CelS. Classical molecular dynamics simulations of CNT-bound CelS through one of the selected binding sites resulted in negligible changes in the secondary structure of the enzyme and its catalytic domain, implying the least possible effect on the catalytic activity post-immobilization. Furthermore, our study reveals that while the unfolding near the CNT-binding region in CelS is more pronounced when the enzyme is interacting with a wider CNT, resulting in enhanced contact area and improved binding affinity, its impact on the overall CelS structure is relatively less significant when compared to thinner CNTs. Particularly, CNTs of diameter ∼12 Šcan serve as a favorable option for substrate materials in cellulase immobilization. Our study also provides critical insights into the binding mechanisms between cellulase and CNTs, which could lead to the development of more efficient biocatalysts for biofuel production.

Structural and Biophysical Characterization of Purified Recombinant Arabidopsis thaliana's Alternative Oxidase 1A (rAtAOX1A): Interaction With Inhibitor(s) and Activator.

In higher plants, alternative oxidase (AOX) participates in a cyanide resistant and non-proton motive electron transport pathway of mitochondria, diverging from the ubiquinone pool. The physiological significance of AOX in biotic/abiotic stress tolerance is well-documented. However, its structural and biophysical properties are poorly understood as its crystal structure is not yet revealed in plants. Also, most of the AOX purification processes resulted in a low yield/inactive/unstable form of native AOX protein. The present study aims to characterize the purified rAtAOX1A protein and its interaction with inhibitors, such as salicylhydroxamic acid (SHAM) and n-propyl gallate (n-PG), as well as pyruvate (activator), using biophysical/in silico studies. The rAtAOX1A expressed in E. coli BL21(DE3) cells was functionally characterized by monitoring the respiratory and growth sensitivity of E. coli/pAtAOX1A and E. coli/pET28a to classical mitochondrial electron transport chain (mETC) inhibitors. The rAtAOX1A, which is purified through affinity chromatography and confirmed by western blotting and MALDI-TOF-TOF studies, showed an oxygen uptake activity of 3.86 µmol min-1 mg-1 protein, which is acceptable in non-thermogenic plants. Circular dichroism (CD) studies of purified rAtAOX1A revealed that >50% of the protein content was α-helical and retained its helical absorbance signal (ellipticity) at a wide range of temperature and pH conditions. Further, interaction with SHAM, n-PG, or pyruvate caused significant changes in its secondary structural elements while retaining its ellipticity. Surface plasmon resonance (SPR) studies revealed that both SHAM and n-PG bind reversibly to rAtAOX1A, while docking studies revealed that they bind to the same hydrophobic groove (Met191, Val192, Met195, Leu196, Phe251, and Phe255), to which Duroquinone (DQ) bind in the AtAOX1A. In contrast, pyruvate binds to a pocket consisting of Cys II (Arg174, Tyr175, Gly176, Cys177, Val232, Ala233, Asn294, and Leu313). Further, the mutational docking studies suggest that (i) the Met195 and Phe255 of AtAOX1A are the potential candidates to bind the inhibitor. Hence, this binding pocket could be a 'potential gateway' for the oxidation-reduction process in AtAOX1A, and (ii) Arg174, Gly176, and Cys177 play an important role in binding to the organic acids like pyruvate.

CelS-Catalyzed Processive Cellulose Degradation and Cellobiose Extraction for the Production of Bioethanol.

Bacterial cellulase enzymes are potent candidates for the efficient production of bioethanol, a promising alternative to fossil fuels, from cellulosic biomass. These enzymes catalyze the breakdown of cellulose in plant biomass into simple sugars and then to bioethanol. In the absence of the enzyme, the cellulosic biomass is recalcitrant to decomposition due to fermentation-resistant lignin and pectin coatings on the cellulose surface, which make them inaccessible for hydrolysis. Cellobiohydrolase CelS is a microbial enzyme that binds to cellulose fiber and efficiently cleaves it into a simple sugar (cellobiose) by a repeated processive chopping mechanism. The two contributing factors to the catalytic reaction rate and the yield of cellobiose are the efficient product expulsion from the product binding site of CelS and the movement of the substrate or cellulose chain into the active site. Despite progress in understanding product expulsion in other cellulases, much remains to be understood about the molecular mechanism of processive action of these enzymes. Here, nonequilibrium molecular dynamics simulations using suitable reaction coordinates are carried out to investigate the energetics and mechanism of the substrate dynamics and product expulsion in CelS. The calculated free energy barrier for the product expulsion is three times lower than that for the processive action indicating that product removal is relatively easier and faster than the sliding of the substrate to the catalytic active site. The water traffic near the active site in response to the product expulsion and the processive action is also explored.

Correlated Response of Protein Side-Chain Fluctuations and Conformational Entropy to Ligand Binding.

The heterogeneous fast side-chain dynamics of proteins plays crucial roles in molecular recognition and binding. Site-specific NMR experiments quantify these motions by measuring the model-free order parameter (Oaxis2) on a scale of 0 (most flexible) to 1 (least flexible) for each methyl-containing residue of proteins. Here, we have examined ligand-induced variations in the fast side-chain dynamics and conformational entropy of calmodulin (CaM) using five different CaM-peptide complexes. Oaxis2 of CaM in the ligand-free (Oaxis,U2) and ligand-bound (Oaxis,B2) states are calculated from molecular dynamics trajectories and conformational energy surfaces obtained using the adaptive biasing force (ABF) method. ΔOaxis2 = Oaxis,B2 - Oaxis,U2 follows a Gaussian-like unimodal distribution whose second moment is a potential indicator of the binding affinity of these complexes. The probability for the binding-induced Oaxis,U2 → Oaxis,B2 transition decreases with increasing magnitude of ΔOaxis2, indicating that large flexibility changes are improbable for side chains of CaM after ligand binding. A linear correlation established between ΔOaxis2 and the conformational entropy change of the protein makes possible the determination of the conformational entropy of binding of protein-ligand complexes. The results not only underscore the functional importance of fast side-chain fluctuations but also highlight key motional and thermodynamic correlates of protein-ligand binding.

Solubility of Caffeine in Supercritical CO2: A Molecular Dynamics Simulation Study.

The extraction of caffeine from green tea leaves and cocoa beans is a common industrial process for the production of decaffeinated beverages and pharmaceuticals. The choice of the solvent critically determines the yield of this extraction process. Being an environmentally benign and recyclable solvent, supercritical carbon dioxide (scCO2) has emerged as the most desirable green solvent for caffeine extraction. The present study investigates the solvation properties of caffeine in scCO2 at two different temperatures (318 and 350 K) using molecular dynamics simulations. Unlike in water, the caffeine molecules in scCO2 do not aggregate to form clusters due to relatively stronger caffeine-CO2 interactions. A well-structured scCO2 solvent shell envelops each caffeine molecule as a result of strong electron-donor-acceptor (EDA) and hydrogen-bonding interactions between these two species. Upon heating, although marginal site-specific changes in the distribution of nearest CO2 around caffeine are observed, the overall distribution is retained. At a higher temperature, the caffeine-CO2 hydrogen-bonding interactions are weakened, while their EDA interactions become relatively stronger. The results underscore the importance of the interplay of these interactions in determining stable solvent structures and solubility of caffeine in scCO2.

Molecular dynamics simulation of protein-mediated biomineralization of amorphous calcium carbonate.

The protein-mediated biomineralization of calcium carbonate (CaCO3) in living organisms is primarily governed by critical interactions between the charged amino acids of the protein, solvent, calcium (Ca2+) and carbonate (CO3 2-) ions. The present article investigates the molecular mechanism of lysozyme-mediated nucleation of amorphous calcium carbonate (ACC) using molecular dynamics and metadynamics simulations. The results reveal that, by acting as nucleation sites, the positively charged side chains of surface-exposed arginine residues form hydrogen bonds with carbonates and promote aggregation of ions around them leading to the formation and growth of ACC on the protein surface. The newly formed ACC patches were found to be less hydrated due to ion aggregation-induced expulsion of water from the nucleation sites. Despite favorable electrostatic interactions of the negatively charged side chains of aspartate and glutamate with calcium ions, these residues contribute minimally to the growth of ACC on protein surface. The activation barrier for the growth of partially hydrated ACC patches on lysozymes was determined from the free energy profiles obtained from metadynamics simulations.

Water dynamics in hydrated amorphous materials: a molecular dynamics study of the effects of dehydration in amorphous calcium carbonate.

Amorphous calcium carbonate (ACC) is often a critical transient phase in the formation of crystalline phases of CaCO3via dehydration of hydrated ACC. The behavior of the water molecules plays a pivotal role in this transformation. We report here the dynamics of water molecules in ACC at hydration levels from CaCO3·1H2O to CaCO3·0.25H2O using molecular dynamics (MD) simulations. Due to the presence of highly hydrophilic Ca2+ and the strong H-bond acceptor CO32-, most of the water molecules in our simulations have restricted translational and orientational dynamics. These are referred here as 'slow water'. However, a small fraction of them show high diffusivity at all hydration states and are referred here as 'fast water'. The computed diffusion coefficients of the slow waters, as extrapolated from simulation results, yield diffusion distances of the order of µm on the 1 hour time scale, consistent with rapid dehydration of ACC nano-particles in the laboratory. We correlate our fast waters with the water molecules in the percolating water-rich, Ca2+-deficient nano-pores in the structure of Goodwin et al. [Chem. Mater., 2010, 22, 3197], obtained by analysis of X-ray scattering data, and our slow waters with those in the Ca2+-rich volumes with less water in their model. The fast waters can be considered to be free rotors on the ns time scale and have orders of magnitude shorter rotational relaxation times than the slow waters.

Ab initio and metadynamics studies on the role of essential functional groups in biomineralization of calcium carbonate and environmental situations.

The interactions of proteins, polysaccharides and other biomolecules with Ca(2+), CO3(2-), and water are central to the understanding of biomineralization and crystallization of calcium carbonate (CaCO3), and their association with the natural organic matter (NOM) in the environment. A molecular-level investigation of how such interactions and thermodynamic forces drive the nucleation and growth of crystalline CaCO3 in living organisms remains elusive. This paper presents ab initio and metadynamics studies of the interactions of Ca(2+), CO3(2-), and water with the essential amino acids/functional groups, e.g. arginine/NH2(+), aspartate or glutamate/COO(-), aspartic or glutamic acid/COOH, and serine/OH, of protein/organic molecules that are likely to be critical to the biomineralization of CaCO3. These functional groups were modeled as guanidinium (Gdm(+)), acetate (AcO(-)), acetic acid (AcOH), and ethanol (EtOH) molecules, respectively. The Gdm(+)-Ca(2+)-CO3(2-) and AcO(-)-Ca(2+)-CO3(2-) systems were found to form stable ion-complexes irrespective of the presence of near neighbor water molecules. The strong electrostatic interactions of these functional groups with their counter-ions significantly affect the fundamental vibrational frequencies of the functional groups, mainly the NH2 stretching (str.) and degenerate (deg.) scissors modes of Gdm(+) and -C=OO, CC, and CO str. modes of AcO(-). The free-energy calculations reveal that EtOH forms weakly bound molecular complexes with the Ca(2+)-CO3(2-) ion pairs in water. However, the interaction strength of EtOH with crystalline CaCO3 can increase significantly due to combined effect of H-bond and electron donor acceptor (EDA) type of interactions. These results indicate that -NH2(+) and -COO(-) bearing molecules serve as potential nucleation sites promoting crystallization of CaCO3 phases while -OH bearing molecules are likely to control the morphology of the crystalline phases by attaching to the growing crystal surfaces.

Dehydration-induced amorphous phases of calcium carbonate.

Amorphous calcium carbonate (ACC) is a critical transient phase in the inorganic precipitation of CaCO3 and in biomineralization. The calcium carbonate crystallization pathway is thought to involve dehydration of more hydrated ACC to less hydrated ACC followed by the formation of anhydrous ACC. We present here computational studies of the transition of a hydrated ACC with a H2O/CaCO3 ratio of 1.0 to anhydrous ACC. During dehydration, ACC undergoes reorganization to a more ordered structure with a significant increase in density. The computed density of anhydrous ACC is similar to that of calcite, the stable crystalline phase. Compared to the crystalline CaCO3 phases, calcite, vaterite, and aragonite, the computed local structure of anhydrous ACC is most-similar to those of calcite and vaterite, but the overall structure is not well described by either. The strong hydrogen bond interaction between the carbonate ions and water molecules plays a crucial role in stabilizing the less hydrated ACC compositions compared to the more hydrated ones, leading to a progressively increasing hydration energy with decreasing water content.

Catalytic mechanism of cellulose degradation by a cellobiohydrolase, CelS.

The hydrolysis of cellulose is the bottleneck in cellulosic ethanol production. The cellobiohydrolase CelS from Clostridium thermocellum catalyzes the hydrolysis of cello-oligosaccharides via inversion of the anomeric carbon. Here, to examine key features of the CelS-catalyzed reaction, QM/MM (SCCDFTB/MM) simulations are performed. The calculated free energy profile for the reaction possesses a 19 kcal/mol barrier. The results confirm the role of active site residue Glu87 as the general acid catalyst in the cleavage reaction and show that Asp255 may act as the general base. A feasible position in the reactant state of the water molecule responsible for nucleophilic attack is identified. Sugar ring distortion as the reaction progresses is quantified. The results provide a computational approach that may complement the experimental design of more efficient enzymes for biofuel production.

Evolution of intermolecular structure and dynamics in supercritical carbon dioxide with pressure: an ab initio molecular dynamics study.

The effect of pressure on supercritical carbon dioxide (scCO2) has been characterized by using Car-Parrinello molecular dynamics simulations. Structural and dynamical properties along an isotherm of 318.15 K and at pressures ranging from 190 to 5000 bar have been obtained. Intermolecular pair correlation functions and three-dimensional atomic probability density map calculations indicate that the local environment of a central CO2 molecule becomes more structured with increasing pressure. The closest neighbors are predominantly oriented in a distorted T-shaped geometry while neighbors separated by larger distances are likely oriented in a slipped parallel arrangement. The structure of scCO2 at high densities has been compared with that of crystalline CO2. The probability distributions of intramolecular distances narrow down with increasing pressure. A marginal but non-negligible effect of pressure on the instantaneous intramolecular OCO angle is observed, lending credence to the idea that intermolecular interactions between CO2 molecules in an inhomogeneous near neighbor environment could contribute to the observed instantaneous molecular dipole moment. The extent of deviation from a perfect linear geometry of the carbon dioxide molecule decreases with increasing pressure. Time constants derived from reorientational time correlation functions of the molecular backbone compare well with experimental data. Within the range of thermodynamic conditions explored here, no significant changes are observed in the frequencies of intramolecular vibrational modes. However, a blue shift is observed in the low-frequency cage rattling mode with increasing pressure.

Electron donor-acceptor interactions in ethanol-CO2 mixtures: an ab initio molecular dynamics study of supercritical carbon dioxide.

The nature of interactions between ethanol and carbon dioxide has been characterized using simulations via the Car-Parrinello molecular dynamics (CPMD) method. Optimized geometries and energetics of free-standing ethanol-CO2 clusters exhibit evidence for a relatively more stable electron donor-acceptor (EDA) complex between these two species rather than a hydrogen-bonded configuration. This fact has also been confirmed by the higher formation rate of the EDA complex in supercritical carbon dioxide-ethanol mixtures. The probability density distribution of CO2 molecules around ethanol in the supercritical state shows two high probability regions along the direction of the lone pairs on the oxygen atom of ethanol. The EDA interaction between ethanol and CO2 as well as that between CO2 molecules themselves leads to significant deviations from linearity in the geometry of the CO2 molecule. The vibrational spectra of carbon dioxide obtained from the atomic velocity correlation functions in the bulk system as well as from isolated complexes show splitting of the nu2 bending mode that arises largely from CO2-CO2 interactions, with ethanol contributing only marginally because of its low concentration in the present study. The stretching frequency of the hydroxyl group of ethanol is shifted to lower frequencies in the bulk mixture when compared to its gas-phase value, in agreement with experiments.

Ab initio molecular-dynamics study of supercritical carbon dioxide.

Car-Parrinello molecular-dynamics simulations of supercritical carbon dioxide (scCO(2)) have been performed at the temperature of 318.15 K and at the density of 0.703 g/cc in order to understand its microscopic structure and dynamics. Atomic pair correlation functions and structure factors have been obtained and good agreement has been found with experiments. In the supercritical state the CO(2) molecule is marginally nonlinear, and thus possesses a dipole moment. Analyses of angle distributions between near neighbor molecules reveal the existence of configurations with pairs of molecules in the distorted T-shaped geometry. The reorientational dynamics of carbon dioxide molecules, investigated through first- and second-order time correlation functions, exhibit time constants of 620 and 268 fs, respectively, in good agreement with nuclear magnetic resonance experiments. The intramolecular vibrations of CO(2) have been examined through an analysis of the velocity autocorrelation function of the atoms. These reveal a red shift in the frequency spectrum relative to that of an isolated molecule, consistent with experiments on scCO(2). The results have also been compared to classical molecular-dynamics calculations employing an empirical potential.