Search

Maintaining the high activity of an enzyme is a fundamental requirement to widen the application of metal–organic frameworks (MOFs) in the biotechnology, biosensor, and biomedicine fields. However,...

The structural basis for the spectral differences between the Fenna-Matthews-Olson (FMO) proteins from Chlorobaculum tepidum (C. tepidum) and Prosthecochloris aestuarii 2K (P. aestuarii) is yet to be fully understood. Mutation-induced perturbation to the exciton structure and the optical spectra of the complex provide a suitable means to investigate the critical role played by the protein scaffold. In this work, we have performed quantum-mechanics/molecular-mechanics calculations over the molecular dynamics simulation trajectories with the polarized protein-specific charge scheme for both wild-type FMOs and two mutants. Our result reveals that a single-point mutation in the vicinity of BChl 6, namely, W183F of C. tepidum, significantly affects the absorption spectrum, resulting in a switch of the absorption spectrum from type 2, for which the 806 nm band is more pronounced than the 815 nm band, to type 1, for which the 815 nm band is pronounced. Our observations agree with the single-point mutation experiments reported by Saer et al. (Biochim. Biophys. Acta, Bioenerg.2017,1858, 288-296) and Khmelnitskiy et al. (J. Phys. Chem. Lett.2018,9, 3378-3386). In contrast, the absorption spectrum of the P. aestuarii experiences the opposite transition (from type 1 to type 2) upon the same mutation. Furthermore, by comparing the contributions of individual pigments to the spectra in the wild type and its mutant, we find that a single-point mutation near BChl 6 not only induces changes in excitation energy of BChl 6 per se but also affects the excitonic structures of the neighboring BChls 5 and 7 through strong interpigment electronic couplings, resulting in a significant change in the absorption spectra.

Multidrug resistance (MDR) remains an obstacle to chemotherapy related with the overexpression of several efflux membrane proteins, and p-glycoprotein (P-gp) is the most studied among them. Thus, continuous investigational efforts are necessary to find valuable MDR reversal agents, and the flavonoid compound glabridin (GBD) seems to be a promising candidate. This study aimed to investigate the potential of GBD against MDR and explore the possible mechanisms. First, we found that GBD could decrease the half maximal inhibitory concentration of paclitaxel and doxorubicin (DOX) in breast cancer cells like MDA-MB-231/MDR1 cells and MCF-7/ADR cells. It was further explained that GBD enhanced the apoptosis of MDA-MB-231/MDR1 cells induced by DOX, due to the increased accumulation of DOX. Then, tests were performed to explore the possible MDR reversal mechanisms. On one hand, GBD can suppress the expression of P-gp. On the other hand, GBD can downregulate the activity of P-gp ATPase when cotreated with DOX or verapamil, revealing that GBD was a substrate of P-gp. Moreover, the obtained kinetic inhibition parameters proved that GBD was a competitive inhibitor of P-gp, and in molecular docking simulation modeling, GBD exhibited stronger binding affinity with P-gp than DOX. In conclusion, GBD can increase the accumulation of DOX in MDA-MB-231/MDR1 cells by suppressing the expression of P-gp and competitively inhibiting the P-gp efflux pump and enhance the apoptosis of MDA-MB-231/MDR1 cells induced by DOX, and thus realize reversal effects on MDR. Therefore, the combination therapy of anticancer drugs and flavonoid-like GBD is a promising strategy to overcome P-gp-mediated MDR.

The microscopic insight into the endo/exo stereoselectivity of the Diels-Alder (DA) reaction between cyclopentadiene and methyl vinyl ketone (MVK) has posed a challenge to the computational chemists, which requires an accurate free-energy (FE) landscape. Although ab initio (ai) quantum mechanical/molecular mechanical (QM/MM) calculations are capable of providing accurate conformation energies, they are far too expensive for free-energy calculations, in which large-scale sampling in the phase space is always indispensable. Recently, on the basis of the idea of reference-potential methods, a new approach termed MBAR+wTP method was proposed by us ( Li ; J. Chem. Theory Comput. 2018 , 14 , 5583 ) for the calculations of ai QM/MM FE profiles with a much less computational expense. In this work, we applied this method to investigate the endo/exo stereoselectivity of the Diels-Alder (DA) reaction at B3LYP/MM level and the results indicate that this method can yield more accurate activation free energies than the semiempirical Hamiltonian PM6 and the "sampling-free" quantum mechanical methods at B3LYP and MP2 levels using a polarizable continuous solvent model (PCM). The stereoselectivity mainly comes from the solvation effect. At the transition state, the first peak of the solvent distribution near the oxygen atom in MVK is slightly closer for the endo pathway than that for the exo pathway, whereas the difference at PM6 level is negligible. Nonetheless, in terms of the stereoselectivity, we obtained an endo/exo ratio of 2 with this method, which is still 1 order of magnitude smaller than the experimental measurement. Although the endo/exo ratio from the MP2/PCM calculation agrees well with the experiment, the absolute reaction barriers deviate by about 5 kcal mol

Graphene quantum dots (GQDs), with stable photoluminescence (PL) and electrocatalytic activity, have potential application in sensor field. Herein, a facile novel electrochemiluminescence (ECL) sensor based on GQDs and glassy carbon electrode (GCE) was developed to detect Butyl hydroxy anisd (BHA) and Gallic acid. The optimization of ECL detection conditions were investigated. Hence, the sensor exhibited relatively strong luminescence intensity even without the modification of the GCE, which make this sensor system easy to implement. The developed sensor exhibited a linear response ranging from 5 to 160 μM under the optimum conditions. The method displayed the advantages of high sensitivity, good selectivity, wide linear range, low detection limit and fine reproducibility. Moreover, the practicability of the ECL sensing platform in real water samples had shown the satisfactory results, which holds a great potential in the field of pharmaceutical analysis.

Osteoporosis is a common metabolic bone disease with high prevalence. Tetrandrine (TET) suppressed osteoclastogenesis, while the roles of TET in osteoporosis regulation remained unclear. Thus, the study aimed to investigate the effect of TET on osteoporosis and the underlying mechanism.The osteoporosis rabbit model was established through anterior cruciate ligament transection (ACLT) and bilateral ovariectomy (OVX). The degeneration of articular cartilage was assessed using HE staining and Alcian blue staining. The liver and kidney tissue injury was determined using HE staining. The activity of osteoclasts was evaluated using Tartrate-resistant acid phosphatase (TRAP) staining. The changes in bone structural parameters were determined through measuring the BMD, BV/TV, Tb.Th, Tb.N, and Tb.Sp, and the serum levels of calcium and phosphorus. Macrophage polarization was determined using Flow cytometry.The bone structural parameters including BMD, BV/TV, Tb.N, Tb.Th and Tb.Sp were changed in osteoporosis rabbit, which was reversed by TET. Besides, TET suppressed the increased serum levels of calcium and phosphorus in osteoporosis rabbit. Furthermore, TET inhibited the degeneration of articular cartilage and the activity of osteoclasts induced by osteoporosis. Moreover, TET inhibited the levels of MMP-9, PPAR-γ, RANKL, β-CTX and TRACP-5b, and increased the levels of OPG, ALP and osteocalcin (OC) in osteoporosis. Additionally, TET promoted macrophage transformation from M1 to M2 in osteoporotic and inhibited the production of IL-1β, TNF-α, and IL-6. TET also inhibited the p65 phosphorylation in osteoporosis. Besides, TET reversed RANKL-induced osteoclasts proliferation, p65 phosphorylation, and the expression changes of RANKL, Ki67, PPAR-γ, ALP, OPG.TET attenuated bone structural parameters including BMD, BV/TV, Tb.N, Tb.Th and Tb.Sp, inhibited articular cartilage degeneration, promoted bone formation, inhibited the inflammatory response, and promoted macrophage transformation from M1 to M2 via NF-κB inactivation in osteoporosis. TET may be a promising drug for osteoporosis therapy.TET: Tetrandrine; ACLT: anterior cruciate ligament transection; OVX: ovariectomy; TRAP: Tartrate-resistant acid phosphatase; BMD: bone mineral density; BV/TV: Bone volume/total volume; Tb.Th: trabecular thickness; Tb.N: trabecular number; Tb.Sp: trabecular separation; MMP-9: Matrix metallopeptidase 9; PPAR-γ: Peroxisome proliferator-activated receptor gamma; RANKL: Receptor activator of nuclear factor kappa-B ligand; OPG: Osteoprotegerin; ALP: alkaline phosphatase; OC: osteocalcin; β-CTX: β isomer of C-terminal telopeptide of type I collagen; TRACP-5b: Tartrate-resistant acid phosphatase 5b; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; IL-6: interleukin 6; NF-κB: Nuclear factor kappa B; PKC-α: Protein kinase C alpha; qRT-PCR: Quantitative real-time polymerase chain reaction.

Aggregation of the misfolded scrapie prion protein (PrPSc) is known to cause neurodegenerative diseases. In this paper, we have investigated the stability of PrPC by combining coarse-grained model and all-atom molecular simulations. Our results show that the unfolding of PrPC starts from the opening of the folded domain with α1 moving away from α2α3 domain, and then arrives at a metastable intermediate, and forms a more stable dimer complex in the end. This work unravels the mechanism of the early stage of conformational conversion and dimerization of prion protein and provides significant hints for the development of anti-prion therapeutics.

A water soluble and stable metal-organic framework (MOF) [Cu(Dcbcp)(bpe)] n ( 1 , Dcbcp = N-(3,5-dicarboxylbenzyl)-(3-carboxyl) pyridinium, bpe = 1,2-bis(4-pyridyl)ethylene) was synthesized. MOF 1 possesses a three-dimensional open framework with large pores and has a high affinity toward carboxyfluorescein (FAM) or 5(6)-carboxyrhodamine, triethylammonium salt (ROX)-tagged single-stranded probe DNA (P-DNA) through π stacking, electrostatic interactions, thus quenching the fluorescence of tag. The two formed P-DNA@ 1 systems can be applied for simultaneous detection of Dengue virus (DENV) and Zika virus (ZIKV) RNA sequences, which is attributed to the different affinities of MOF 1 with P-DNA and the double-stranded DNA/RNA formed upon recognition. With the same detection time of 36 min and 2 min, the detection limits are 332 and 192 pM with the single detection method, 184 and 121 pM with synchronous fluorescence detection method. Both assays are highly specific and not interfered by other mismatched RNA sequences, even down to single-base mismatched RNA sequences. There was no cross-reaction between the two probes for synchronous detection. Comparing these two methods, synchronous fluorescence analysis improves the detection efficiency by saving the time and increasing the detection limits, which may attribute to its avoiding the interference of Raleigh light scattering signal to the fluorescence signal.

The selectivities in C–H oxidations of a variety of compounds by DMDO have been explored with density functional theory. There is a linear Evans–Polanyi-type correlation for saturated substrates. Activation energies correlate with reaction energies or, equivalently, BDEs (ΔH‡sat = 0.91*BDE – 67.8). Unsaturated compounds, such as alkenes, aromatics, and carbonyls, exhibit a different correlation for allylic and benzylic C–H bonds (ΔH‡unsat = 0.35*BDE – 13.1). Bernasconi’s Principle of Non-Perfect Synchronization (NPS) is found to operate here. The origins of this phenomenon were analyzed by a Distortion/Interaction model. Computations indicate early transition states for H-abstractions from allylic and benzylic C–H bonds, but later transition states for the saturated. The reactivities are mainly modulated by the distortion energy and the degree of dissociation of the C–H bond. While the increase in barrier with higher BDE is not unexpected from the Evans−Polanyi relationship, two separate correlations, one...

Intrinsically disordered proteins (IDPs) carry out crucial biological functions in essential biological processes of life. Because of the highly dynamic and conformationally heterogeneous nature of the disordered states of IDPs, molecular dynamics simulations are becoming an indispensable tool for the investigation of the conformational ensembles and dynamic properties of IDPs. Nevertheless, there is still no consensus on the most reliable force field in molecular dynamics simulations for IDPs hitherto. In this work, the recently proposed AMBER99SB2D force field is evaluated in modeling some disordered polypeptides and proteins by checking its ability to reproduce experimental NMR data. The results highlight that when the ildn side-chain corrections are included, AMBER99SB2D-ildn exhibits reliable results that agree with experiments compared with its predecessors, the AMBER14SB, AMBER99SB, AMBER99SB-ildn, and AMBER99SB2D force fields, and that decreasing the overall magnitude of protein–protein interactio...

Using the FTIR-ATR technique, we investigated the heterogeneous nucleation process of aqueous (NH 4 ) 2 SO 4 binary droplets and (NH 4 ) 2 SO 4 /glycerol ternary droplets. From the red shift of δ -NH 4 + with a linearly declining relative humidity (RH), the ERHs of pure (NH 4 ) 2 SO 4 droplets and mixed (NH 4 ) 2 SO 4 /glycerol droplets with different organic-inorganic ratio (OIR) of 1:4, 1:2 and 1:1 ranges from ∼51.9 to ∼34.9%, ∼49.8 to ∼33.0%, ∼48.0 to ∼30.6% and ∼43.7 to ∼25.2%, respectively. From the changing absorbance of δ -NH 4 + band, we determined the heterogeneous nucleation rates of crystalline (NH 4 ) 2 SO 4 in the pure and mixed droplets. The interfacial tension between an (NH 4 ) 2 SO 4 critical nucleus and surrounding (NH 4 ) 2 SO 4 solution was determined using classical nucleation theory and experimental data to be 0.031 ± 0.002 J m −2 . Evaporation of glycerol in (NH 4 ) 2 SO 4 /glycerol ternary droplets are also studied to be restrained by participation of (NH 4 ) 2 SO 4 . Determined vapour pressure of glycerol is on the same magnitude with results from previous studies.

In this work, we report two polarizable molecular mechanics (polMM) force field models for estimating the polarization energy in hybrid quantum mechanical molecular mechanical (QM/MM) calculations. These two models, named the potential of atomic charges (PAC) and potential of atomic dipoles (PAD), are formulated from the ab initio quantum mechanical (QM) response kernels for the prediction of the QM density response to an external molecular mechanical (MM) environment (as described by external point charges). The PAC model is similar to fluctuating charge (FQ) models because the energy depends on external electrostatic potential values at QM atomic sites; the PAD energy depends on external electrostatic field values at QM atomic sites, resembling induced dipole (ID) models. To demonstrate their uses, we apply the PAC and PAD models to twelve small molecules, which are solvated by TIP3P water. The PAC model reproduces the QM/MM polarization energy with a R2 value of 0.71 for aniline (in 10,000 TIP3P water configurations) and 0.87 or higher for other eleven solute molecules, while the PAD model has a much better performance with R2 values of 0.98 or higher. The PAC model reproduces reference QM/MM hydration free energies for twelve solute molecules with a RMSD of 0.59 kcal/mol. The PAD model is even more accurate, with a much smaller RMSD of 0.12 kcal/mol, with respect to the reference. This suggests that polarization effects, including both local charge distortion and intramolecular charge transfer, can be well captured by induced dipole type models with proper parameterization.

A method that can reliably predict protein–ligand binding free energies is essential for rational drug design. Much effort has been devoted to this field, but it remains challenging especially for flexible ligands. In this work, both a molecular mechanical (MM) method and a hybrid quantum mechanical/molecular mechanical (QM/MM) method have been applied in the study of the binding affinities of methyl-α-L-fucoside to Ralstonia solanacearum lectins. The free energy at the MM level was calculated using the double-decoupling method (DDM) and the free energy change at each step was calculated via a series of intermediate states using the Bennett acceptance ratio (BAR). The binding free energy agrees well with the experimental measurement, no matter whether the general AMBER force field or GLYCAM06j was applied to the ligand. Nonetheless, slow convergence for some intermediate states has been observed, which requires substantially longer simulations than were used in many other studies. The QM/MM free energy was calculated by thermodynamic perturbation (TP) from the MM states. This strategy has been shown to yield minimal variance for the calculated free energy without direct sampling at the QM/MM level in a previous study. However, after this MM-to-QM/MM correction, the agreement with the experimental value decreased. This study serves as an implication of the demand for substantially longer simulations for the alchemical process than those that were used in many other studies and for further improvement of QM/MM methods, especially the description of interactions between the QM and MM regions.

Human herpesvirus 6B (HHV-6B) belongs to the β-herpesvirus subfamily of the Herpesviridae. To understand capsid assembly and capsid-tegument interactions, here we report atomic structures of HHV-6B capsid and capsid-associated tegument complex (CATC) obtained by cryoEM and sub-particle reconstruction. Compared to other β-herpesviruses, HHV-6B exhibits high similarity in capsid structure but organizational differences in its CATC (pU11 tetramer). 180 “VΛ”-shaped CATCs are observed in HHV-6B, distinguishing from the 255 “Λ”-shaped dimeric CATCs observed in murine cytomegalovirus and the 310 “Δ”-shaped CATCs in human cytomegalovirus. This trend in CATC quantity correlates with the increasing genomes sizes of these β-herpesviruses. Incompatible distances revealed by the atomic structures rationalize the lack of CATC’s binding to triplexes Ta, Tc, and Tf in HHV-6B. Our results offer insights into HHV-6B capsid assembly and the roles of its tegument proteins, including not only the β-herpesvirus-specific pU11 and pU14, but also those conserved across all subfamilies of Herpesviridae., Human Herpesvirus 6B (HHV-6) can cause fever, diarrhea and roseola rash. Here the authors present a cryoEM approach to image crude, minimally purified virus samples and employ it to determine the atomic structures of HHV-6B capsid and capsid-associated tegument protein complexes.

Bletilla striata has been widely used as a valuable hemostatic agent for thousands of years due to the high levels of bioactive constituents it contains. Here, we used a sensitive ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) method for the simultaneous determination of three major active ingredients of the B. striata extract, namely, α-isobutylmalic acid, gymnoside I, and militarine in rat plasma. The three major active ingredients were determined using the multiple reaction monitoring (MRM) mode at m/z 189 ⟶ 129 for α-isobutylmalic acid, m/z 457.2 ⟶ 285.1 for gymnoside I, m/z 725.3 ⟶ 457.2 for militarine, and m/z 417.0 ⟶ 267.0 for the IS puerarin. All calibration curves showed good linearity (R2 ≥ 0.999) over the concentration range with the lower limit of quantification between 0.015 and 0.029 μg/mL. The relative standard deviations of intraday and interday measurements were less than 15%, and the method was accurate within 93.3–100.4%. The extraction recovery was 92.65–100.98%, and no matrix effect was observed. The results indicated that after oral administration of B. striata in rats, the Tmax of α-isobutylmalic acid was significantly longer than that of gymnoside I and militarine and the mean residence time and area under the curve of α-isobutylmalic acid and gymnoside I in rats were significantly higher than those of militarine. Moreover, the blood concentration-time curve of α-isobutylmalic acid showed double peaks, suggesting that α-isobutylmalic acid could exhibit the phenomenon of enterohepatic circulation or metabolic conversion. We also explored some of the pharmacokinetic characteristics of three ingredients from B. striata extract in vivo, and the data obtained may provide a basis for the further investigation of B. striata.

Neurons undergo degeneration, apoptosis and death due to ischaemic stroke. The present study investigated the effect of Sijunzi decoction (SJZD), a type of traditional Chinese medicine known as invigorating spleen therapy, on anoikis (a type of apoptosis) in rat brains following cerebral ischaemia-reperfusion. Rats were randomly divided into sham, model, nimodipine and SJZD low/medium/high dose groups. A middle cerebral artery occlusion model was established. Neurobehavioural scores were evaluated after administration for 14 days using a five-grade scale. Blood-brain barrier permeability and apoptotic rate were detected using Evans blue (EB) extravasation and TUNEL staining, respectively. Tissue inhibitor of metalloproteinase 1 (TIMP-1), matrix metalloproteinase 9 (MMP-9) and collagen IV (COL IV) were determined using immunohistochemistry. Neurobehavioural scores decreased remarkably in all SJZD and nimodipine groups compared to the model group (P

3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a crucial role in the signal transduction of massive growth-related protein kinases. In this work, a computational study has been performed to investigate the binding pose of the hydrolyzed product of SBF1 (SBF1-) with PDK1. The binding pose was predicted by Vina and was further refined in a molecular dynamics simulation. For comparison, four published low molecular weight compounds (PS48, PS171, PS182, and PS210) binding with PDK1 were also studied. SBF1- was anchored in the PIF-pocket of PDK1 with salt bridge interaction using its carboxylate moiety, which is a common feature among the known ligands. Hydrogen bonds to THR148 and vdW interactions with GLN150 also have contributions to the association affinity. The allosteric regulation on PDK1 via the binding of SBF1- was further addressed. The binding affinity of SBF1- in complex with PDK1 is comparable to those of PS171 and PS182, with an estimated IC50 in a range from 2.0 to 10.0 μ molar. Comparison between the free energy profiles with the presence or absence of SBF1- in the binding pocket indicates that the binding of SBF1- enhances the hinge motion and suppresses the fluctuation of the end-to-end distance in α B of PDK1. These results demonstrate that SBF1- is a promising allosteric regulator of PDK1 targeting the PIF-binding pocket and can serve as a new scaffold template for the design of new drugs targeting PDK1.

Relative free energies for the binding of nine cyclic carboxylate ligands to the octa-acid deep-cavity host were calculated at the combined density-functional theory and molecular mechanics (DFT/MM) level of theory. The DFT calculations employed the BLYP functional and the 6-31G* basis set for the ligand. We employed free-energy perturbations (FEP) with the reference-potential approach and used molecular dynamics (MD) simulations with the semiempirical quantum mechanical (SQM) PM6-DH+ method for the ligand as an intermediate level between MM and DFT/MM to improve the convergence. Thus, the relative binding free energy of two ligands was first calculated at the MM level by an alchemical transformation from one ligand to another in both the bound and unbound states. Then, for each ligand the free-energy correction for going from the MM to the SQM/MM potentials was calculated using explicit SQM/MM MD simulations. Finally, the free-energy correction for going from the SQM/MM to the DFT/MM potentials was estimated with FEP without running any DFT/MM simulations. Instead, the free energy was calculated by single-step exponential averaging (ssEA) or employing the cumulant approximation to the second order (CA). The results show that CA converges much better than ssEA, and with 500-4500 DFT/MM single-point energy calculations, converged free energies with a precision of 0.3 kJ/mol can be obtained. These free energies reproduce the experimental binding free energy differences with a mean absolute deviation of 3.4 kJ/mol, a correlation ( R2) of 0.97, and correct signs for all of the eight free-energy differences. This is appreciably better than the results obtained at the SQM/MM level of theory and also slightly better than those obtained with MM. We show that the convergence of the SQM/MM → DFT/MM perturbations can be monitored by the use of Wu and Kofke's bias metric Π and by the standard deviation of the difference between the SQM/MM and DFT/MM energies. Finally, we show that the use of the intermediate SQM/MM MD simulations improves the convergence of the free energies by a factor of at least two, compared to doing direct MM → DFT/MM perturbations.

Cytomegalovirus (CMV) infection causes birth defects and life-threatening complications in immunosuppressed patients. Lack of vaccine and need for more effective drugs have driven widespread ongoing therapeutic development efforts against human CMV (HCMV), mostly using murine CMV (MCMV) as the model system for preclinical animal tests. The recent publication (Yu et al., 2017, DOI: 10.1126/science.aam6892) of an atomic model for HCMV capsid with associated tegument protein pp150 has infused impetus for rational design of novel vaccines and drugs, but the absence of high-resolution structural data on MCMV remains a significant knowledge gap in such development efforts. Here, by cryoEM with sub-particle reconstruction method, we have obtained the first atomic structure of MCMV capsid with associated pp150. Surprisingly, the capsid-binding patterns of pp150 differ between HCMV and MCMV despite their highly similar capsid structures. In MCMV, pp150 is absent on triplex Tc and exists as a “Λ”-shaped dimer on other triplexes, leading to only 260 groups of two pp150 subunits per capsid in contrast to 320 groups of three pp150 subunits each in a “Δ”-shaped fortifying configuration. Many more amino acids contribute to pp150-pp150 interactions in MCMV than in HCMV, making MCMV pp150 dimer inflexible thus incompatible to instigate triplex Tc-binding as observed in HCMV. While pp150 is essential in HCMV, our pp150-deletion mutant of MCMV remained viable though with attenuated infectivity and exhibiting defects in retaining viral genome. These results thus invalidate targeting pp150, but lend support to targeting capsid proteins, when using MCMV as a model for HCMV pathogenesis and therapeutic studies., Author summary Cytomegalovirus (CMV) infection is a leading viral cause of birth defects and could be deadly to AIDS patients and organ transplant recipients. Absence of effective vaccines and potent drugs against human CMV (HCMV) infections has motivated animal-based studies, mostly based on the mouse model with murine CMV (MCMV), both for understanding pathogenesis of CMV infections and for developing therapeutic strategies. Distinct from other medically important herpesviruses (those responsible for cold sores, genital herpes, shingles and several human cancers), CMV contains an abundant phosphoprotein, pp150, which is a structurally, immunogenically, and regulatorily important tegument protein and a potential drug target. Here, we used cryoEM with localized reconstruction method to obtain the first atomic structure of MCMV. The structure reveals that the organization patterns of the capsid-associated tegument protein pp150 are different in MCMV and HCMV, despite their highly similar capsid structures. We also show that deleting pp150 did not eliminate MCMV infection in contrast to pp150’s essential role in HCMV infections. Our results have significant implication to the current practice of using mouse infected with MCMV for HCMV therapeutic development.

Hygroscopicity is capable of significantly affecting atmospheric visibility, radiative forcing, and cloud condensation nuclei process. Herein, a physisorption analyzer was adopted to determine the hygroscopicity of inorganic salt particles in atmospheric aerosols (e.g., NaNO3, NaClO4, MgSO4, K3PO4, K2HPO4, and KH2PO4) at 298 K. By regulating the ambient relative humidity (RH), qualitative and quantitative information of the mentioned particles in humidifying and dehumidifying cycles was acquired. The deliquescence relative humidity (DRH) and efflorescence relative humidity (ERH) were measured, and supersaturation was observed and recorded during the dehumidification process. The water-to-salt molar ratios (WSR) of the mentioned inorganic salts depending on RH were determined, and the hygroscopic results of NaNO3 and NaClO4 particles were well consistent with the results achieved with other methods or by theoretical prediction. This study fitted WSR-to-RH equations suitable for NaNO3, NaClO4, MgSO4, K3PO4, and K2HPO4 particles. The adsorption heats for monolayer water molecules in the mentioned inorganic salt particles are calculated. The present study lays a solid foundation for gaining insights into the effects of RH on atmospheric aerosols.

In this work, the solvation free energies of 20 organic molecules from the 4th Statistical Assessment of the Modeling of Proteins and Ligands (SAMPL4) have been calculated. The sampling of phase space is carried out at a molecular mechanical level, and the associated free energy changes are estimated using the Bennett Acceptance Ratio (BAR). Then the quantum mechanical (QM) corrections are computed through the indirect Non-Boltzmann Bennett's acceptance ratio (NBB) or the thermodynamics perturbation (TP) method. We show that BAR+TP gives a minimum analytic variance for the calculated solvation free energy at the Gaussian limit and performs slightly better than NBB in practice. Furthermore, the expense of the QM calculations in TP is only half of that in NBB. We also show that defining the biasing potential as the difference of the solute-solvent interaction energy, instead of the total energy, can converge the calculated solvation free energies much faster but possibly to different values. Based on the experimental solvation free energies which have been published before, it is discovered in this study that BLYP yields better results than MP2 and some other later functionals such as B3LYP, M06-2X, and ωB97X-D.

For Diels&ndash, Alder (DA) reactions in solution, an accurate and converged free energy (FE) surface at ab initio (ai) quantum mechanical/molecular mechanical (QM/MM) level is imperative for the understanding of reaction mechanism. However, this computation is still far too expensive. In a previous work, we proposed a new method termed MBAR+wTP, with which the computation of the ai FE profile can be accelerated by several orders of magnitude via a three-step procedure: (I) an umbrella sampling (US) using a semi-empirical (SE) QM/MM Hamiltonian is performed, (II) the FE profile is generated using the Multistate Bennett Acceptance Ratio (MBAR) analysis, and (III) a weighted Thermodynamic Perturbation (wTP) from the SE Hamiltonian to the ai Hamiltonian is performed to obtain the ai QM/MM FE profile using weight factors from the MBAR analysis. In this work, this method is extended to the calculations of two-dimensional FE surfaces of two Diels&ndash, Alder reactions of cyclopentadiene with either acrylonitrile or 1-4-naphthoquinone at ai QM/MM level. The accurate activation free energies at the ai QM/MM level, which are much closer to the experimental measurements than those calculated by other methods, indicate that this MBAR+wTP method can be applied in the studies of complex reactions in condensed phase with much-enhanced efficiency.

A new adsorbent was synthesized from sawdust, a forest residue, in which methanol was used as a solvent and triethylamine as a modification agent under the following optimum conditions: 25 °C of reaction temperature, 1 : 8.75 of the ratio of sawdust to triethylamine (g:mL) and 1 hour of reaction time. The adsorption capacity of this adsorbent for malachite green was improved by 632.98% in contrast to that of the unmodified sawdust under the same adsorption conditions. Factors affecting the adsorption behavior of this adsorbent for malachite green, such as pH value, adsorption time, temperature and initial dye concentration, were evaluated through experiments in a batch system. The results indicated that the maximum adsorption capacity can be achieved at 5.08 of pH value and adsorption equilibrium can be reached in 6 hours. It was also found that the higher the temperature, the higher the adsorptive capacity would be. The Freundlich isotherm model provides a better description for the adsorption equilibrium when compared with the Langmuir equation in the conditions of the present study. Additionally, to examine the controlling mechanisms of the process, kinetic equations of the mass transfer and chemical reaction, the pseudo-first order model, the pseudo-second order model and the intraparticle diffusion model were used to correlate the experimental data respectively. The adsorption process of malachite green on sawdust tended to be controlled simultaneously by film mass transfer and intra-particle diffusion and it accompanied chemical reactions. It showed that the sawdust modified with triethylamine had good performance for cationic dye and can be used as a biomass adsorbent to treat dyes-containing wastewater with high quality.

The conformational change of poly(vinyl alcohol) has been studied by Fourier transform infrared spectroscopy at various temperatures in the 4000–400 cm−1 region. The molecular motion and the trans/gauche content are sensitive to the C H, C C stretching modes. FTIR spectra show that the I2920/I2849 decreases from 1.84 to 1.0 with increasing temperature, companying the decrease in I1047/I1095 from 0.78 to 0.58, implying the conformational transition from trans to gauche in alkyl chain. Based on the van’t Hoff relation, the enthalpies and entropies have been calculated in different temperatures, which are 4.61 kJ mol−1 and 15.23 J mol−1 K−1, respectively, in the region of 80–140 °C. From the C O stretching mode and O H band, it can be concluded that the intermolecular hydrogen bonds decrease owing to elevating temperature, which leads to more gauche conformers.

The partitioning of solute molecules between immiscible solvents with significantly different polarities is of great importance. The polarization between the solute and solvent molecules plays an essential role in determining the solubility of the solute, which makes computational studies utilizing molecular mechanics (MM) rather difficult. In contrast, quantum mechanics (QM) can provide more reliable predictions. In this work, the partition coefficients of the side chain analogs of some amino acids between water and chloroform were computed. The QM solvation free energies were calculated indirectly via a series of MM states using the multistate Bennett acceptance ratio (MBAR) and the MM-to-QM corrections were applied at the two endpoints using thermodynamic perturbation (TP). Previously, it has been shown (Jia et al. J. Chem. Theory Comput. 2016, 12, 499-511) that this method provides the minimal variance in the results without running QM simulations. However, if there is insufficient overlap in phase space between the MM and QM Hamiltonians, this method fails. In this work, we propose, for the first time, a quantity termed the reweighting entropy that serves as a metric for the reliability of the TP calculations. If the reweighting entropy is below a certain threshold (0.65 for the solvation free energy calculations in this work), this MM-to-QM correction should be avoided and two alternative methods can be employed by either introducing a semiempirical state or conducting nonequilibrium simulations. However, the results show that the QM methods are not guaranteed to yield better results than the MM methods. Further improvement of the QM methods are imperative, especially the treatment of the van der Waals and the electrostatic interactions between the QM region and the MM region in the first shell. We also propose a scheme for the calculation of the van der Waals parameters for the solute molecules in nonaqueous solvent, which improves the quality of the computed thermodynamic properties. Furthermore, the force field parameters for the sulfur-containing molecules are also optimized.

MD simulation study of several peptides including a polyalanine, a helix (pdb:2I9M), and a leucine zipper were carried out to investigate hydrogen bond energetics using dynamic polarized protein-specific charge (DPPC) to account for the polarization effect in protein dynamics. Results show that the backbone hydrogen-bond strength is generally correlated with its specific local electrostatic environment, measured by the number of water molecules near the hydrogen bond in the first solvation shell. The correlation coefficient is found to be 0.89, 0.78, and 0.80, respectively, for polyalanine, 2I9M protein, and leucine zipper. In the polyalanine, the energies of the backbone hydrogen bonds are very similar to each other due to their similar local electrostatic environment. The current study helps demonstrate and support the understanding that hydrogen bonds are stronger in a hydrophobic surrounding than in a hydrophilic one. For comparison, the result from simulation using standard force field shows a much weaker correlation between hydrogen bond energy and local electrostatic environment due to the lack of polarization effect in the force field.

In combined quantum mechanical/molecular mechanical (QM/MM) free energy calculations, it is often advantageous to have a frozen geometry for the quantum mechanical (QM) region. For such multiple-environment single-system (MESS) cases, two schemes are proposed here for estimating the polarization energy: the first scheme, termed MESS-E, involves a Roothaan step extrapolation of the self-consistent field (SCF) energy; whereas the other scheme, termed MESS-H, employs a Newton-Raphson correction using an approximate inverse electronic Hessian of the QM region (which is constructed only once). Both schemes are extremely efficient, because the expensive Fock updates and SCF iterations in standard QM/MM calculations are completely avoided at each configuration. They produce reasonably accurate QM/MM polarization energies: MESS-E can predict the polarization energy within 0.25 kcal/mol in terms of the mean signed error for two of our test cases, solvated methanol and solvated β-alanine, using the M06-2X or ωB97X-D functionals; MESS-H can reproduce the polarization energy within 0.2 kcal/mol for these two cases and for the oxyluciferin-luciferase complex, if the approximate inverse electronic Hessians are constructed with sufficient accuracy.

The correct representation of solute-water interactions is essential for the accurate simulation of most biological phenomena. Several highly accurate quantum methods are available to deal with solvation by using both implicit and explicit solvents. So far, however, most evaluations of those methods were based on a single conformation, which neglects solute entropy. Here, we present the first test of a novel approach to determine hydration free energies that uses molecular mechanics (MM) to sample phase space and quantum mechanics (QM) to evaluate the potential energies. Free energies are determined by using re-weighting with the Non-Boltzmann Bennett (NBB) method. In this context, the method is referred to as QM-NBB. Based on snapshots from MM sampling and accounting for their correct Boltzmann weight, it is possible to obtain hydration free energies that incorporate the effect of solute entropy. We evaluate the performance of several QM implicit solvent models, as well as explicit solvent QM/MM for the blind subset of the SAMPL4 hydration free energy challenge. While classical free energy simulations with molecular dynamics give root mean square deviations (RMSD) of 2.8 and 2.3 kcal/mol, the hybrid approach yields an improved RMSD of 1.6 kcal/mol. By selecting an appropriate functional and basis set, the RMSD can be reduced to 1 kcal/mol for calculations based on a single conformation. Results for a selected set of challenging molecules imply that this RMSD can be further reduced by using NBB to reweight MM trajectories with the SMD implicit solvent model.

The reliability of the linear interaction energy (LIE) depends on the atomic charge model used to delineate the Coulomb interaction between the ligand and its environment. In this work, the polarized protein-specific charge (PPC) implementing a recently proposed fitting scheme has been examined in the LIE calculations of the binding affinities for avidin and β-secretase binding complexes. This charge fitting scheme, termed delta restrained electrostatic potential, bypasses the prevalent numerical difficulty of rank deficiency in electrostatic-potential-based charge fitting methods via a dual-step fitting strategy. A remarkable consistency between the predicted binding affinities and the experimental measurement has been observed. This work serves as a direct evidence of PPC's applicability in rational drug design. © 2014 Wiley Periodicals, Inc.

The FTIR-ATR spectra of Na 2 SO 4 and mixed Na 2 SO 4 /glycerol (1:1) aerosols are monitored in the dehumidifying and humidifying process. It shows that glycerol suppresses both heterogeneous and homogeneous nucleation of Na 2 SO 4 /glycerol aerosols on the ZnSe substrate, while the water uptake of surrounding glycerol leads to lower deliquescence relative humidity of mixed Na 2 SO 4 /glycerol aerosols. In mixed Na 2 SO 4 /glycerol aerosols, glycerol restrains the formation of contact ion pairs between Na + and SO 4 2− , resulting in the delay of the efflorescence relative humidity. And glycerol molecular conformations transform from γγ to αα type in the dehumidifying process.

MD simulations of five proteins in which helical chains are held together by hydrophobic packing were carried out to investigate the effect of hydrophobic force on simulated structures of these protein complexes in implicit generalized Born (GB) model. The simulation study employed three different methods to treat hydrophobic effect: the standard GB method that does not include explicit hydrophobic force, the LCPO method that includes explicit hydrophobic force based directly on solvent accessible surface area (SASA), and a proposed packing enforced GB (PEGB) method that includes explicit hydrophobic force based on the radius of gyration of the protein complex. Our simulation study showed that all five protein complexes were unpacked in the standard GB simulation (without explicit hydrophobic force). In the LCPO method, three of the five protein systems remained well packed during the simulation, indicating the need for an explicit hydrophobic force in GB model for these packed protein systems. However, two of the five systems were still unpacked during LCPO simulation. For comparison, all five protein systems remain well packed in simulation using the new PEGB method. Analysis shows that the failure of the LCPO method in two cases is related to the way that SASA changes during the unpacking process for these two systems. These examples showed that standard GB method without explicit hydrophobic force is not suitable for MD simulation of protein systems involving hydrophobic packing. A similar problem remains but to a much lesser extent in the LCPO method for some packed protein systems. The proposed PEGB method seems quite promising for MD simulation of large, multi-domain packed proteins in implicit solvent model.

Elucidating the relationship between sequence and conformation is essential for the understanding of functions of proteins. While sharing 88 % sequence identity and differing by only seven residues, GA88 and GB88 have completely different structures and serve as ideal systems for investigating the relationship between sequence and function. Benefiting from the continuous advancement of the computational ability of modern computers, molecular dynamics (MD) simulation is now playing an increasingly important role in the study of proteins. However, the reliability of MD simulations is limited by the accuracy of the force fields and solvent model approximations. In this work, several AMBER force fields (AMBER03, AMBER99SB, AMBER12SB, AMBER14SB, AMBER96) and solvent models (TIP3P, IGB5, IGB7, IGB8) have been employed in the simulations of GA88 and GB88. The statistical results from 19 simulations show that GA88 and GB88 both adopt more compact structures than the native structures. GB88 is more stable than GA88 regardless of the force fields and solvent models utilized. Most of the simulations overestimated the salt bridge interaction. The combination of AMBER14SB force field and IGB8 solvent model shows the best overall performance in the simulations of both GA88 and GB88. AMBER03 and AMBER12SB also yield reasonable results but only in the TIP3P explicit solvent model.

Previous experimental study measuring the binding affinities of biotin to the wild type streptavidin (WT) and three mutants (S45A, D128A and S45A/D128A double mutant) has shown that the loss of binding affinity from the double mutation is larger than the direct sum of those from two single mutations. The origin of this cooperativity has been investigated in this work through molecular dynamics simulations and the end-state free energy method using the polarized protein-specific charge. The results show that this cooperativity comes from both the enthalpy and entropy contributions. The former contribution mainly comes from the alternations of solvation free energy. Decomposition analysis shows that the mutated residues nearly have no contributions to the cooperativity. Instead, N49 and S88, which are located at the entry of the binding pocket and interact with the carboxyl group of biotin, make the dominant contribution among all the residues in the first binding shell around biotin.

A QM/QM' direct molecular dynamics study of a water-accelerated Diels-Alder reaction in aqueous solution is reported. Cyclopentadiene and methyl vinyl ketone are known to react faster in water than in nonpolar solvents. We have explored how polarization of water molecules afforded by PM3 influences the nature of the transition state, and the reaction dynamics. We compare the results with previous studies on QM/MM and QM/MM+3QM water simulations from our laboratory. Transition state sampling in vacuum PM3 water boxes indicates that the asynchronicity is 0.54 Å in QM/QM', as compared to 0.48 Å in QM/MM, and 0.54 Å in QM/MM+3QM water. The mean time gap between the formation of two C-C bonds is 19 fs for QM/QM', compared to 20 fs for QM/MM, and 25 fs for QM/MM+3QM water. The samplings and time gaps are qualitatively consistent, indicating that water polarization is not significant in sampling and dynamics of bonding changes. The dynamics of hydrogen bonding between reacting molecules and water molecules was also analyzed. From reactants to transition states, H-bond shortening is 0.4 Å by QM/QM', while only 0.15 Å for QM/MM and QM/MM+3QM water. From reactants to transition states, the mean value of the H-bond angle increases by 19° in QM/QM', but only 4° in QM/MM, and 10° in QM/MM+3QM water. These suggest that water polarization is essential for the correct representation of dynamical formation of hydrogen bonds in the transition state by water reorientation. QM/QM' overestimates the hydrogen bonding enhancement because of its underestimation of neutral hydrogen bonding within the reactants, a general deficiency of PM3.

Inspired by the idea of charge decomposition in calculation of the dipole preserving and polarization consistent charges (Zhang et al., J. Comput. Chem. 2011, 32, 2127), we have proposed a numerically stable restrained electrostatic potential (ESP)-based charge fitting method for protein. The atomic charge is composed of two parts. The dominant part is fixed to a predefined value (e.g., AMBER charge), and the residual part is to be determined by restrained fitting to residual ESP on grid points around the molecule. Nonuniform weighting factors as a function of the dominant charge are assigned to the atoms. Because the residual part is several folds to several orders smaller than the dominant part, the impact of ill-conditioning is alleviated. This charge fitting method can be used in quantum mechanical/molecular mechanical (QM/MM) simulations and similar studies, where QM calculated electronic properties are frequently mapped to partial atomic charges.

Cytochrome P450 (CYP) 2J2 is one of the human CYPs involved in phase I xenobiotics metabolism. It is mainly expressed in extrahepatic tissues, including intestine and cardiovascular systems. The general role of CYP2J2 in drug metabolism is not yet fully understood, and the recent discovery that CYP2J2 can metabolize a wide range of structurally diverse drugs and its primary distribution in the intestine suggest its potentially indispensable role in first-pass intestinal metabolism and involvement in drug-drug interaction. To fully characterize its role in drug metabolism, selective and potent inhibitors of CYP2J2 are necessary tools. In the current study, 69 known drugs were screened for the inhibition of CYP2J2, and we discovered a number of marketed drugs as potent and selective CYP2J2 inhibitors. In particular, telmisartan and flunarizine have CYP2J2 inhibition IC(50) values of 0.42 μM and 0.94 μM, respectively, which are at least 10-fold more selective against all other major metabolizing CYPs; moreover, they are not substrates of CYP2J2 and show no time-dependent inhibition toward this CYP. The results of enzyme kinetics studies, supported by molecular modeling, have also elucidated that telmisartan is a mixed-type inhibitor, and flunarizine competitively inhibits CYP2J2. The K(i) for telmisartan is 0.19 μM, with an α value, an indicator of the type of inhibition mechanism, of 2.80, and flunarizine has a K(i) value of 0.13 μM. These newly discovered CYP2J2 inhibitors can be potentially used as a tool to study CYP2J2 in drug metabolism and interaction in a clinical setting.

Conformational changes of the antiparallel β-sheet in normal cellular prion protein (PrPC) of rat, bovine, and human are investigated by molecular dynamics simulations in both neutral and acidic environment. Using a recently developed simulation method based on an on-the-fly polarized protein-specific charge (PPC) update scheme during the simulation process, we evaluate and compare the cross-species performances of the β-sheet during the early stage transition from the PrPC to its mutant configuration. Through this study, we observe the growth of the β-sheet structure in all species studied with the extent of elongation in β-sheet being different across the three species.

We study the dynamical properties of the hydration water around apo-myoglobin in this work under traditional AMBER force field and the polarized protein-specific charges (PPC). Both SPC/E and TIP3P water models are utilized in order to reveal the model dependence. With polarization effect taken into consideration as in the PPC, the hydrogen bonds between the protein and the solvation water molecules are strengthened. Therefore the lifetime of the hydrogen bonds increases, and both the rotational and translational motions of the water molecules are hindered. We also notice that these results have apparent model dependence, when comparing the SPC/E and TIP3P water models.

In this study, we apply both pairwise AMBER03 force field and the recently developed polarized force field to study the folding process of EK peptide under various ion strength and pH conditions. The polarized force field is based on our newly proposed adaptive hydrogen bond-specific charge (AHBC) scheme. These two force fields differ only by the atomic charges. Solvent effect is described with generalized Born models (IGB5 in AMBER 10 package). The result shows that although when applying AMBER03 charge, the helical structure is preferred, its dependence on salt concentration and pH is qualitatively wrong. While using AHBC the peptide finds its native structure within 10 ns, and then fluctuates around this folded state. Under high salt concentration or extreme pH conditions the calculated helical structure probability drops, which is in qualitative agreement with the experiment. Analysis of the atomic charges and the interaction between the donor-acceptor pair in main hydrogen bonds shows that the helical structure is stabilized when polarization effect is counted. It again shows that polarization effect is a very important improvement over traditional force field and is essential for protein folding. We also prove that the salt bridge interaction between 4-residue apart GLU and LYS residues is not critical to the stability of helical structure of EK peptide, but is merely an auxiliary factor, also in agreement with the experiment.

Four kinds of water-soluble polysaccharides A1(from fruit body), A2(from mycelia), PA(purification polysaccharide extracted from fruit body) and A6(from fermentation broth), which were isolated from an edible mushroom Pholiota.adiposa (Fr ) Quel and its aqueous extract, fruit body power and mycelia power of the P. adiposa were studied in antioxidation experiment. They were all found to have the ability of scavenging free radicals, but their effect on O2-• and •OH were different. A6 had the best oxyradical scavenging activity and PA was the best in scavenging hydroxyl radical.

During the summers of years 2005,2006 and 2007,68 species of commercial aquatic products were collected from Mindong ecological monitoring area,Fujian east coast,P.R of China,and the residues of hexachlorobenzene (BHCs ) and dichloro-diphenyl-trichloroethane (DDTs ) were quantified by gas chromatography.The residues levels,distribution patterns and compositions of BHCs and DDTs,as well as the pollution status and the risk to human health were also evaluated and discussed.The results showed that,the residues of BHCs in shell-fish,shrimps,crabs and fishes ranged from undetected to 1.53 μg/kg,undetected to 0.830 μg/kg,undetected to 1.21 μg/kg,undetected to 4.02 μg/kg,in wet weight respectively; and the residues of DDTs ranged from 0.184 to 66.9 μg/kg,undetected to 2.87 μg/kg,8.04 to 126 μg/kg,0.891 to 310 μg/kg,in wet weight respectively.BHCs pollution index was below 0.5,which indicated that commercial aquatic products along Mindong ecological monitoring area had not been polluted by BHCs.71.2% of tested samples with the DDTs pollution index range from 0.581 to 31.0,which indicated they had been more or less polluted by DDTs.BHCs levels in organisms from Mindong ecological monitoring area were lower compared with those reported in other sea waters at home and abroad.DDTs residues were in the middle levels compared with those reported in domestic adjacent waters and were relatively higher compared with those reported in Asia-Pacific region waters.The average ratios of p,p'-DDT/DDTs in shellfishes and fishes were 43.8% and 49.1% respectively.High percentage of p,p'-DDT content may suggest that some contaminants such as DDTs had input into the Mindong ecological monitoring area in recent years.The residues levels of BHCs and DDTs were below the Maximum Residual Limits published by the European Union,USA and Japan; and met the sea food safety index set by Chinese government.The risk of organochlorine pesticides to human health was assessed according to RfD( reference dose) ,and cancer slope factor suggested by EPA( US EPA,2000) .The analyzing of ERI( exposure risk index) and CRI( carcinogenic risk index) indicated that the exposure risk and carcinogenic potential arising from the exposure to BHCs and DDTs due to the consumption of aquatic product in Mindong ecological monitoring zone were within acceptable ranges.

Docking of the drug raltegravir to HIV-1 integrase (IN) was performed based on the established Relaxed Complex Scheme (RCS) method which accounts for the flexibility of both receptor and ligand in molecular docking. Two representative butterfly-like structures of raltegravir were identified and both of them mimicked the binding mode of 5CITEP with similar ligand-receptor interactions. Furthermore, the results that raltegravir interacted with magnesium by intermediate water molecules indicate the importance of water molecules at the binding site which has always been ignored in the docking studies of IN inhibitors. Taking these water molecules into consideration gives more insight into the design and development of the second generation IN inhibitors.

We report direct folding of a 17-residue helix protein (pdb:2I9M) by standard molecular dynamics simulation (single trajectory) at room temperature with implicit solvent. Starting from a fully extended structure, 2I9M successfully folds into the native conformation within 16 ns using adaptive hydrogen bond-specific charges to take into account the electrostatic polarization effect. Cluster analysis shows that conformations in the native state cluster have the highest population (78.4%) among all sampled conformations. Folding snapshots and the secondary structure analysis demonstrate that the folding of 2I9M begins at terminals and progresses toward the center. A plot of the free energy landscape indicates that there is no significant free energy barrier during folding, which explains the observed fast folding speed. For comparison, exactly the same molecular dynamics simulation but carried out under existing AMBER charges failed to fold 2I9M into native-like structures. The current study demonstrates that electrostatic polarization of intraprotein hydrogen bonding, which stabilizes the helix, is critical to the successful folding of 2I9m.

Three novel schemes on the basis of newly developed polarized protein-specific charge (PPC) have been proposed to refine the structure of staphylococcal nuclease generated by AMBER simulation. The performance of the refinement is analyzed in term of the stability and the recovery of intra-protein hydrogen bonds.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2mM Mn(2+). The RT retained in the column was eluted after soaking overnight in 10mM EDTA to retrieve the Mn(2+). In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs.

Molecular dynamics simulations based on AMBER force fields (ff96 and ff03) and generalized Born models (igb1 and igb5) have been carried out in order to study folding/unfolding of the Trp-cage mini-protein TC5b. The thermodynamic properties of TC5b were found to be sensitive to the specific version of the solvation model and force field employed. When the ff96/igb5 combination was used, the predicted melting temperature from unfolding simulations was in good agreement with the experimental value of 315 K, but the folding simulation did not converge. The most stable thermodynamic profile in both folding and unfolding simulations was obtained when the ff03/igb5 combination was employed, and the predicted melting temperature was about 345 K, showing over-stabilization of the protein. Simulations using the igb1 version in combination with ff96 or ff03 were difficult to converge within the simulation time limit (50 ns).