A conformational approach to the study of the dynamics of enzyme inhibition: studies on thermolysin

The preferred conformations of β-phenylpropionyl-l-phenylalanine (β-PPP) and $\text{N-}\text{carbobenzoxy-}\text{l}\text{-phenylalanine}$ (Cb_z-Phe), two inhibitors of thermolysin, have been determined by computing potential energy using empirial potential energy functions. Of the 15 to 20 conformations that are favoured for each of these inhibitors only a few have the right conformation to reach the active site of the enzyme. The conformer of β-PPP that initiates binding with the enzyme is different from the bound one, while for Cb_z-Phe the bound and initiating conformers are quite similar. Thus, β-PPP favours the ‘induced fit’ model while Cb_z-Phe follows the ‘lock and key’ model of binding. The inhibitors differ in their alignment at the active site.     

Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin

Site-directed mutagenesis of porcine pepsin was performed to identify its active sites that regulate nucleic acid (NA) digestion activity and to analyze the mechanism pepsin-mediated NA digestion. The mutation sites were distributed at the catalytic center of the enzyme (T33A, G34A, Y75H, T77A, Y189H, V214A, G217A and S219A) and at its active site (D32A and D215A) for protein digestion. Mutation of the active site residues Asp32 and Asp215 led to the inactivation of pepsin (both the NA and protein digestion activity), which demonstrated that the active sites of the pepsin protease activity were also important for its nuclease activity. Analysis of the variants revealed that T33A and G217A mutants showed a complete loss of NA digestion activity. In conclusion, residues Asp32, Thr33, Asp215 and Gly217 were related to the pepsin active sites for NA digestion. Moreover, the Y189H and V214A variants showed a loss of digestion activity on double-strand DNA (dsDNA) but only a decrease in digestion activity on single-strand DNA (ssDNA). On the contrary, the G34A variant showed a loss of digestion activity on ssDNA but only a decrease in digestion activity on dsDNA. Our findings are the first to identify the active sites of pepsin nuclease activity and lay the framework for further study of the mechanism of pepsin nuclease activity.     

Soil Contamination at Dumpsites: Implication of Soil Heavy Metals Distribution in Municipal Solid Waste Disposal System: A Case Study of Abeokuta,Southwestern Nigeria

Soil distribution of heavy metals caused by municipal solid waste (MSW) deposition and its implications for MSW management system in emerging cities was investigated in Abeokuta, Nigeria. Results indicated that the highest concentrations of Cu, Cr, Mn, and Zn were observed at 0-40 cm while Pb, Fe, and Ni accumulated at depths below 40 cm. Soils affected by waste deposits from market and auto-mechanic sites showed high levels of Fe, Cr, Pb, Cu, Mn, and Zn. The accumulation of heavy metals in the soils was probably due to the formation of metal-organo-complexes. Therefore, source separation of MSW with proper management systems is proposed to improve the indiscriminate surface dumping practiced at present, while the use of wastes affected sites for cultivation should be discouraged.     

The transitional ER defines a boundary for quality control in the secretion of tsO45 VSV glycoprotein

Quality control in the secretory pathway limits forward transport of newly synthesized cargo proteins to those that have acquired their fully folded conformation. To determine which organelles participate in this conformation-dependent sorting process, we analyzed the trafficking of the temperature-sensitive, thermo-reversible folding mutant of vesicular stomatitis virus glycoprotein (tsO45 G protein) in VERO cells. Using temperature blocks, the G protein could be localized to the ER (39.5 °C), to the vesiculo-tubular clusters (VTCs, 15 °C), and to the trans- Golgi network (TGN, 20 °C). To localize the G protein specifically to ER exit sites, we incubated cells at 10 °C. The exit sites contained Sec13p, a COPII component, and were devoid of calnexin and other ER chaperones. We found that if the G protein in the exit sites was misfolded by a temperature shift from 10 °C to 39.5 °C, it failed to enter the VTCs. Instead, it was returned to the reticular ER where it associated with calnexin. However, if the G protein was in the VTCs or beyond, its folding status no longer affected further transport. The observations indicate that quality control took place in the ER and in the ER transitional elements, but not in the VTCs or the Golgi complex. The results provide a way to discriminate biochemically between exit sites and VTCs, two related structures that are difficult to distinguish from each other.     

Effect of CaCN2 on the preparation of CaAlSiN3:Eu2+ phosphors

Highly efficient red‐emitting phosphors, CaAlSiN₃:Eu²⁺, were successfully prepared by the solid‐state method using calcium cyanide (CaCN₂) as the single calcium source. The influences of crystallization temperature, crystallization time, calcination mode and compounds ratio on the photoluminescent properties were investigated. The CaAlSiN₃:Eu²⁺ phosphors were obtained with 1 mol% CaCN₂ by a two‐step calcination procedure at 900°C for 2 h and subsequently at 1600°C for 8 h. The CaAlSiN₃:Eu²⁺ phosphors showed the strongest luminescent intensity at 660 nm when excited by 468 nm. With an increase in crystallization time, the maximum wavelength of the emission was shifted from 644 nm to 660 nm.     

鸭源H9N2AIV血凝素基因序列比较

为明确国内外鸭源H9N2亚型禽流感病毒(Avian influenza virus,AIV)血凝素基因(hemagglutinin,HA)的遗传进化关系、血凝素蛋白裂解位点的氨基酸结构特征和血凝素蛋白受体结合位点的氨基酸变异特征,本研究选取GenBank中登录鸭源H9N2亚型AIV HA基因,通过MEGA4.1进行比对和分析,并绘制其遗传进化树。结果表明,鸭源H9N2亚型AIV在遗传进化上分为2大谱系:即Ck-Bj-1-94-like和North-Ame-like,中国大陆鸭源H9N2亚型AIV和亚欧美其它国家鸭源H9N2亚型AIV在遗传进化上分居完全不同的谱系,相互之间遗传进化关系较远。从血凝素受体结合位点看,亚欧美国家鸭源H9N2亚型AIV在第183、190和226位点的氨基酸均为鸭源AIV经典H、E和Q,且高度保守。但中国大陆地区H9N2亚型AIV第183位为N;第190位为A or V or T,与中国大陆鸡源H9N2亚型AIV一致;第226位中国鸭源H9N2亚型AIV有相当一部分为L,且近年福建省H9N2亚型AIV分离株在此处均为L。提示我们,中国大陆地区H9N2亚型AIV鸭鸡和鸡鸭相互交叉感染较为普遍。     

Molecular simulation study of the quantum effects of hydrogen adsorption in metal-organic frameworks: influences of pore size and temperature

In this work a systematic molecular simulation study was performed to investigate the influence of pore size and temperature on the quantum effects of hydrogen adsorption in metal-organic frameworks (MOFs) with temperature varied from 40 to 120 K. To do this, three isoreticular MOFs (IRMOFs) with different pore sizes were adopted, and quadratic Feynman–Hibbs (FH) effective potential was introduced to consider the quantum effect. The results show that quantum effects diminish with increasing pore size of IRMOFs at lower pressure (loading), while the opposite trend appears at higher pressure (loading). Through the simulations it is also found that the quantum effects may be dominantly determined by the adsorbate–adsorbate or adsorbate–MOFs interactions with the varying of pressure (loading). In addition, the results also indicate how the temperature influences the quantum effects of H₂ adsorption in MOFs within the pressure range considered.     

Solid-state circular dichroism and hydrogen bonding: absolute configuration of massarigenin A from Microsphaeropsis sp

Massarigenin A (1) and papyracillic acids A (2) and B (3) were isolated from the endophytic fungus Microsphaeropsis sp. Their structures were elucidated by multidimensional nuclear magnetic resonance spectroscopy; the structure of massarigenin A (1) was also confirmed by X-ray crystallography. The absolute configuration of massarigenin A (1) was established by means of circular dichroism (CD) spectroscopy and time-dependent density functional theory (TDDFT) calculations. The impact of intermolecular hydrogen bonds detected in the crystal packing of 1 on CD spectra measured in the solid state was also investigated.     

Analysis of the overdispersed clock in the short-term evolution of hepatitis C virus: Using the E1/E2 gene sequences to infer infection dates in a single source outbreak

The assumption of a molecular clock for dating events from sequence information is often frustrated by the presence of heterogeneity among evolutionary rates due, among other factors, to positively selected sites. In this work, our goal is to explore methods to estimate infection dates from sequence analysis. One such method, based on site stripping for clock detection, was proposed to unravel the clocklike molecular evolution in sequences showing high variability of evolutionary rates and in the presence of positive selection. Other alternatives imply accommodating heterogeneity in evolutionary rates at various levels, without eliminating any information from the data. Here we present the analysis of a data set of hepatitis C virus (HCV) sequences from 24 patients infected by a single individual with known dates of infection. We first used a simple criterion of relative substitution rate for site removal prior to a regression analysis. Time was regressed on maximum likelihood pairwise evolutionary distances between the sequences sampled from the source individual and infected patients. We show that it is indeed the fastest evolving sites that disturb the molecular clock and that these sites correspond to positively selected codons. The high computational efficiency of the regression analysis allowed us to compare the site-stripping scheme with random removal of sites. We demonstrate that removing the fast-evolving sites significantly increases the accuracy of estimation of infection times based on a single substitution rate. However, the time-of-infection estimations improved substantially when a more sophisticated and computationally demanding Bayesian method was used. This method was used with the same data set but keeping all the sequence positions in the analysis. Consequently, despite the distortion introduced by positive selection on evolutionary rates, it is possible to obtain quite accurate estimates of infection dates, a result of especial relevance for molecular epidemiology studies.     

A computational method to characterize the missense mutations in the catalytic domain of GAA protein causing Pompe disease

Pompe disease is an autosomal recessive lysosomal storage disease caused by acid α-glucosidase (GAA) deficiency, resulting in intralysosomal accumulation of glycogen, including cardiac, skeletal, and smooth muscle cells. The GAA gene is located on chromosome 17 (17q25.3), the GAA protein consists of 952 amino acids; of which 378 amino acids (347-726) falls within the catalytic domain of the protein and comprises of active sites (518 and 521) and binding sites (404, 600, 616, and 674). In this study, we used several computational tools to classify the missense mutations in the catalytic domain of GAA for their pathogenicity and stability. Eight missense mutations (R437C, G478R, N573H, Y575S, G605D, V642D, L705P, and L712P) were predicted to be pathogenic and destabilizing to the protein structure. These mutations were further subjected to phenotyping analysis using SNPeffect 4.0 to predict the chaperone binding sites and structural stability of the protein. The mutations R437C and G478R were found to compromise the chaperone-binding activity with GAA. Molecular docking analysis revealed that the G478R mutation to be more significant and hinders binding to the DNJ (Miglustat) compared with the R437C. Further molecular dynamic analysis for the two mutations demonstrated that the G478R mutation was acquired higher deviation, fluctuation, and lower compactness with decreased intramolecular hydrogen bonds compared to the mutant R437C. These data are expected to serve as a platform for drug design against Pompe disease and will serve as an ultimate tool for variant classification and interpretations.     

Radiotherapy for the treatment of pituitary adenomas: A dosimetric comparison of three planning techniques

AimOur goal was to compare conformal 3D (C3D) radiotherapy (RT), modulated intensity RT (IMRT), and volumetric modulated arc therapy (VMAT) planning techniques in treating pituitary adenomas.BackgroundRT is important for managing pituitary adenomas. Treatment planning advances allow for higher radiation dosing with less risk of affecting organs at risk (OAR).Materials and methodsWe conducted a 5-year retrospective review of patients with pituitary adenoma treated with external beam radiation therapy (C3D with flattening filter, flattening filter-free [FFF], IMRT, and VMAT). We compared dose-volume histogram data. For OARs, we recorded D2%, maximum, and mean doses. For planning target volume (PTV), we registered V95%, V107%, D95%, D98%, D50%, D2%, minimum dose, conformity index (CI), and homogeneity index (HI).ResultsFifty-eight patients with pituitary adenoma were included. Target-volume coverage was acceptable for all techniques. The HI values were 0.06, IMRT; 0.07, VMAT; 0.08, C3D; and 0.09, C3D FFF (p < 0.0001). VMAT and IMRT provided the best target volume conformity (CI, 0.64 and 0.74, respectively; p < 0.0001). VMAT yielded the lowest doses to the optic pathway, lens, and cochlea. The position of the neck in extreme flexion showed that it helps in planning mainly with VMAT by allowing only one arc to be used and achieving the desired conformity, decreasing the treatment time, while allowing greater protection to the organs of risk using C3D, C3DFFF.ConclusionsOur results confirmed that EBRT in pituitary adenomas using IMRT, VMAT, C3D, C3FFF provide adequate coverage to the target. VMAT with a single arc or incomplete arc had a better compliance with desired dosimetric goals, such as target coverage and normal structures dose constraints, as well as shorter treatment time. Neck extreme flexion may have benefits in treatment planning for better preservation of organs at risk. C3D with extreme neck flexion is an appropriate treatment option when other treatment techniques are not available.     

Calcium binding to the transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase: insights from molecular modeling

Sarcoplasmic reticulum Ca(2+)- ATPase pumps Ca(2+) ions from muscle cells to the sarcoplasmic reticulum. Here we use molecular dynamics and electrostatic modeling to investigate structural and dynamical features of key intermediates in the Ca(2+) binding process of the protein. Structural models of the protein (containing either two, one, or no calcium ions in the transmembrane domain) are constructed based on the X-ray structure by Toyoshima et al. (Nature 2000;405:647-655). The protein is embedded in a water/octane bilayer, which mimics the water/membrane environment. Our calculations provide information on the hydration of the two Ca(2+) ions, not emerging from the X-ray structure. Furthermore, they indicate that uptake of the metal ions causes large structural rearrangements of the metal binding sites. In addition, they suggest that the two ions reach their binding sites via two specific pathways. Finally, they allow identification of residues in the outer mouth of the protein that might interact with the Ca(2+) ions during the binding process.     

A simple method for the simulation of steady-state diffusion through membranes: pressure-tuned,boundary-driven molecular dynamics

We present a novel molecular dynamics-based simulation technique for investigating gas transport through membranes. In our simulations, the main control parameters are the partial pressure for the components on the input side of the membrane and the total pressure on the output side. The essential point of our scheme is that this pressure control should be realised by adjusting the particle numbers in the input and output side control cells indirectly. Although this perturbation is applied sufficiently far from the membrane, the bulk-phase properties are well controlled in a simulation cell of common size. Numerical results are given for silicalite-1 membrane with permeating CH₄, CO₂, H₂ and N₂ gases as well as with binary mixtures of CO₂ with the other three components. To describe interactions between particles, we used the simple shifted and cut Lennard–Jones potential with parameters available in the literature. It is expected that the proposed technique can be applied to several other types of membranes and transported fluids in order to support the development of a deeper understanding of separation processes.     

Mechanochemical Synthesis: A Tool to Tune Cation Site Disorder and Ionic Transport Properties of Li3MCl6 (M = Y,Er) Superionic Conductors

The lithium‐conducting, rare‐earth halides, Li₃MX₆ (M = Y, Er; X = Cl, Br), have garnered significantly rising interest recently, as they have been reported to have oxidative stability and high ionic conductivities. However, while a multitude of materials exhibit a superionic conductivity close to 1 mS cm^?1, the exact design strategies to further improve the ionic transport properties have not been established yet. Here, the influence of the employed synthesis method of mechanochemical milling, compared to subsequent crystallization routines as well as classic solid‐state syntheses on the structure and resulting transport behavior of Li₃ErCl₆ and Li₃YCl₆ are explored. Using a combination of X‐ray diffraction, pair distribution function analysis, density functional theory, and impedance spectroscopy, insights into the average and local structural features that influence the underlying transport are provided. The existence of a cation defect within the structure in which Er/Y are disordered to a new position strongly benefits the transport properties. A synthetically tuned, increasing degree of this disordering leads to a decreasing activation energy and increasing ionic conductivity. This work sheds light on the possible synthesis strategies and helps to systematically understand and further improve the properties of this class of materials.     

A principal component analysis of the dynamics of subdomains and binding sites in human serum albumin

The conformational dynamics of human serum albumin (HSA) was investigated by principal component analysis (PCA) applied to three molecular dynamics trajectories of 200 ns each. The overlap of the essential subspaces spanned by the first 10 principal components (PC) of different trajectories was about 0.3 showing that the PCA based on a trajectory length of 200 ns is not completely convergent for this protein. The contributions of the relative motion of subdomains and of the subdomains (internal) distortion to the first 10 PCs were found to be comparable. Based on the distribution of the first 3 PC, 10 protein conformers are identified showing relative root mean square deviations (RMSD) between 2.3 and 4.6 Å. The main PCs are found to be delocalized over the whole protein structure indicating that the motions of different protein subdomains are coupled. This coupling is considered as being related to the allosteric effects observed upon ligand binding to HSA. On the other hand, the first PC of one of the three trajectories describes a conformational transition of the protein domain I that is close to that experimentally observed upon myristate binding. This is a theoretical support for the older hypothesis stating that changes of the protein onformation favorable to binding can precede the ligand complexation. A detailed all atoms PCA performed on the primary Sites 1 and 2 confirms the multiconformational character of the HSA binding sites as well as the significant coupling of their motions. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 561–572, 2014.     

Hydrolysis of the amide bond in methionine-containing peptides catalyzed by various palladium(II) complexes: Dependence of the hydrolysis rate on the steric bulk of the catalyst

¹H NMR spectroscopy was applied to study the reactions of cis-[Pd(L)(H₂O)₂]²⁺ complexes (L is en, pic and dpa) with the N-acetylated tripeptides L-methionylglycylglycine, MeCOMet–Gly–Gly, and glycyl–L-methionyl–glycine, MeCOGly–Met–Gly. All reactions were performed in the pH range 2.0–2.5 with equimolar amounts of the cis-[Pd(L)(H₂O)₂]²⁺ complex and the tripeptide at 60 °C. The hydrolytic reactions of the cis-[Pd(en)(H₂O)₂]²⁺, cis-[Pd(pic)(H₂O)₂]²⁺ and cis-[Pd(dpa)(H₂O)₂]²⁺ complexes with MeCOMet–Gly–Gly were regioselective and only the amide bond involving the carboxylic group of methionine was cleaved. However, in the reactions of these three Pd(II) complexes with MeCOGly–Met–Gly, two amide bonds, Met–Gly and MeCO–Gly, were cleaved. From UV–Vis spectrophotometry studies, it was found that the rate-determining step of these hydrolytic reactions is the monodentate coordination of the corresponding Pd(II) complex to the sulfur atom of the methionine side chain. The rate of the cleavage of these amide bonds is dependent on the nature of the bidentate coordinated diamine ligand L (en > pic > dpa). The hydrolytic reaction of cis-[Pd(L)(H₂O)₂]²⁺-type complexes with MeCOMet–Gly–Gly, containing the methionine side chain in the terminal position of the peptide, is regioselective while in the reaction of these Pd(II) complexes with MeCOGly–Met–Gly, none selective cleavage of the peptide occurs. This study contributes to a better understanding of the selective cleavage of methionine-containing peptides employing palladium(II) complexes as catalysts.     

A Life Cycle Assessment on the Dehairing of Rawhides: Chemical Treatment versus Enzymatic Recovery through Solid State Fermentation

The leather industry needs to switch from the traditional chemically based dehairing process to an environmentally friendly one so that the overall burdens to the environment are reduced. The primary goal of the work was thus to compare the chemical leather dehairing process to an enzymatically based one using the enzymes that are extracted after the application of solid state fermentation (SSF) on hair wastes generated after dehairing. The environmental burdens of the dehairing stage were determined using a life cycle assessment (LCA) approach by comparing the two aforementioned management scenarios. The first scenario was the commonly used technology in which hair is removed via a chemical process and then composted in open piles. This scenario included two subscenarios where hair waste is either incinerated or landfilled. In the second scenario, the proteolytic enzymes extracted during the SSF of the residual hair are used to dehair the new rawhides instead of chemicals. Industrial and laboratory data were combined with international databases using the SimaPro 8.0 LCA software to make comparisons. The environmental impacts associated with the enzymatic dehairing were significantly lower than the ones associated to the conventional chemical dehairing process. This difference is attributed to the impacts associated with the original production of the chemicals and to the electricity consumed in the conventional method. A sensitivity analysis revealed that the results are affected by the amounts of chemicals used during dehairing.     

5-Enolpyruvylshikimate-3-phosphate synthase: determination of the protonation state of active site residues by the semiempirical method

EPSP synthase (EPSPS) catalyzes the addition of shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form a tetrahedral intermediate (TI) that is converted to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. A semiempirical molecular modeling study of the EPSPS active site containing the TI was implemented for the assignment of the protonation states of four basic residues, Lys22, Lys340, His385, and Lys411, based on the evaluation of 16 different protonation states and comparison of the resulting energy minimized heavy atoms coordinates with available X-ray crystallographic data of the D313A mutant of EPSPS. The results, employing both gas phase and continuum solvent models, are indicative that after the TI formation the histidine residue is most probably in neutral form (N^ε-protonated) and the lysine residues are in protonated form, which suggests that none of the presently proposed assignments of aminoacid residues involved in the reaction mechanism could be completely correct. The protonated state of Lys22 in the presence of the TI supports the proposal that this residue is a general acid catalyst for TI breakdown. Modeling of the native enzyme active site suggests that Asp313 residue has only minor effects on the definition of the TI position inside the active site. Hydrogen-bonds distances suggest that, in order to act as a base, Asp313 needs the intermediacy of a hydroxyl group of the TI for effecting the attack on the TI methyl group in the elimination step leading to EPSP, as suggested previously in the literature.     

A comparison of the electromechanical properties of structurally diverse proteins by molecular dynamics simulation

Proteins are subjected to electric fields both within the cell and during routine biochemical analysis. We have used atomistic molecular dynamics simulations to study conformational changes within three structurally diverse proteins subjected to high electric fields. At electric fields in excess of .5?V/nm, major structural changes were observed in all three proteins due to charge redistribution within the biomolecule. However, the electromechanical resilience was found to be highly dependent on the protein secondary structure, with α-helices showing a particularly high susceptibility to deformation by the applied electric field.     

A common feature-based 3D-pharmacophore model generation and virtual screening: identification of potential PfDHFR inhibitors

A four-feature 3D-pharmacophore model was built from a set of 24 compounds whose activities were reported against the V1/S strain of the Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme. This is an enzyme harboring Asn51Ile + Cys59Arg + Ser108Asn + Ile164Leu mutations. The HipHop module of the Catalyst program was used to generate the model. Selection of the best model among the 10 hypotheses generated by HipHop was carried out based on rank and best-fit values or alignments of the training set compounds onto a particular hypothesis. The best model (hypo1) consisted of two H-bond donors, one hydrophobic aromatic, and one hydrophobic aliphatic features. Hypo1 was used as a query to virtually screen Maybridge2004 and NCI2000 databases. The hits obtained from the search were subsequently subjected to FlexX and Glide docking studies. Based on the binding scores and interactions in the active site of quadruple-mutant PfDHFR, a set of nine hits were identified as potential inhibitors.