Development of SAAP3D force field and the application to replica-exchange Monte Carlo simulation for chignolin and C-peptide.

Single amino acid potential (SAAP) would be a prominent factor to determine peptide conformations. To prove this hypothesis, we previously developed SAAP force field for molecular simulation of polypeptides. In this study, the force field was renovated to SAAP3D force field by applying more accurate three-dimensional main-chain parameters, instead of the original two-dimensional ones, for the amino acids having a long side-chain. To demonstrate effectiveness of the SAAP3D force field, replica-exchange Monte Carlo (REMC) simulation was performed for two benchmark short peptides, chignolin (H-GYDPETGTWG-OH) and C-peptide (CHO-AETAAAKFLRAHA-NH2). For chignolin, REMC/SAAP3D simulation correctly produced native ß-turn structures, whose minimal all-atom root-mean-square deviation value measured from the native NMR structure (except for H) was 1.2 Å, at 300 K in implicit water, along with misfolded ß-hairpin structures with unpacked aromatic side chains of Tyr2 and Trp9. Similar results were obtained for chignolin analog [G1Y,G10Y], which folded more tightly to the native ß-turn structure than chignolin did. For C-peptide, on the other hand, the α-helix content was larger than the ß content on average, suggesting a significant helix-forming propensity. When the imidazole side chain of His12 was protonated (i.e., [His12Hip]), the α content became larger. These observations as well as the representative structures obtained by clustering analysis were in reasonable agreement not only with the structures of C-peptide that were determined in this study by NMR in 30% CD3CD in H2O at 298 K but also with the experimental and theoretical behaviors having been reported for protonated C-peptide. Thus, accuracy of the SAAP force field was improved by applying three-dimensional main-chain parameters, supporting prominent importance of SAAP for peptide conformations.

Model study using designed selenopeptides on the importance of the catalytic triad for the antioxidative functions of glutathione peroxidase.

Although the catalytic triad of glutathione peroxidase (GPx) has been well recognized, there was little evidence for the relevance of the interactions among the triad amino acid residues, i.e., selenocysteine (U), glutamine (Q), and tryptophan (W), to the GPx antioxidative functions. Using a designed selenopeptide having an amino acid sequence of GQAUAWG, we demonstrate here that U, Q, and W present at the active site can interact with each other to exert the enzymatic activity. The amino acid sequence was chosen on the basis of the Monte Carlo molecular simulation for various selenopeptides in polarizable continuous water using the SAAP force field (SAAP-MC). Measurement of the GPx-like activity for the selenopeptide obtained by solid-phase peptide synthesis revealed that the antioxidant activity is cooperatively enhanced by the presence of Q and W proximate to U, although the activity was low compared to selenocystine (U2). The effect of Q on the activity was more important than that of W. In addition, the fluorescence spectrometry suggested a close contact between U and W. These experimental observations were supported by SAAP-MC simulation as well as by ab initio calculation. The latter further suggested that the interaction mode among the triad changes depending on the intermediate states.

Dipeptide inhibitors of thermolysin and angiotensin I-converting enzyme.

Thermolysin (TLN) and other thermolysin-like zinc metalloproteinases (TLPs),are important virulence factors for pathogenesis of bacterial infections by suppressing the innate immune system of the host. Therapeutic inhibition ofTLPs is believed to be a novel strategy inthe development of a new generation antibiotics.In the present study inhibition of TLN and angiotensin I-converting enzyme (ACE) by small peptides were studied by in vitro binding assays and theoretical calculations. The capacity of the peptides to inhibitTLN induced cleavage ofthe transcription factor nuclear factor kappa beta (NF-κB) was studied by electrophoretic mobility shift assays (EMSAs).Nine peptides inhibited ACE with IC50 values in the range 0.48 (IVY) to 1408 (HF) µM, while seven inhibited TLN with IC50 values in the range 0.00034 (IY) to 95640 (FW) µM. Calculations indicated that the peptides occupied the S1' and S2' subsites of ACE, and that IY, LW and IW occupiedthe S1' and S2' subsites, while FW, WL and WV occupiedthe S1 and S1' subsites of TLN. EMSA showed that peptides inhibited TLN induced cleavage of NF-κB. The studied peptides may form as a basis for the design of new compoundstargeting TLN with a potential in the treatment of bacterial infections.

Structure-based analysis of the molecular recognitions between HIV-1 TAR-RNA and transcription factor nuclear factor-kappaB (NFkB).

In this paper we applied the "macromolecular docking" procedure to perform molecular modeling with the aim of screening transcription factor sequences for possible interaction to the HIV-1 TAR-RNA, employing the software Hex version 4.2. The molecular modeling data were compared with electrophoretic mobility shift assays (EMSA) and surface plasmon resonance (SPR) based biospecific interaction analysis (BIA) using an optical biosensor. Finally the specific interactions between NF-κB and RNA have been calculated utilizing the AMBER-MM and FMO calculations. The results obtained clearly indicate that (a) NF-kB p50 transcription factor can bind TAR-RNA; (b) this binding efficiency is lower than that displayed by NF-kB factor in respect to DNA sequences; (c) other structured RNAs used as controls do not bind to NF-kB; (d) TAR-RNA is capable to bind pre-formed NF-kB/DNA complexes. Despite the fact that our data do not indicate whether NF-kB/TAR-RNA complexes play a role in the early steps of HIV-1 transcriptional activation, the results presented strongly indicate that interactions between transcription factors recruited at the level of HIV-1 LTR might interact with the TAR-RNA and deserve further studies aimed to determine its role in the HIV-1 life cycle.

Specific interactions and binding energies between thermolysin and potent inhibitors: molecular simulations based on ab initio molecular orbital method.

Biochemical functions of the metalloprotease thermolysin (TLN) are controlled by various inhibitors. In a recent study we identified 12 compounds as TLN inhibitors by virtual screening and in vitro competitive binding assays. However, the specific interactions between TLN and these inhibitors have not been clarified. We here investigate stable structures of the solvated TLN-inhibitor complexes by classical molecular mechanics simulations and elucidate the specific interactions between TLN and these inhibitors at an electronic level by using ab initio fragment molecular orbital (FMO) calculations. The calculated binding energies between TLN and the inhibitors are qualitatively consistent with the experimental results, and the FMO results elucidate important amino acid residues of TLN for inhibitor binding. Based on the calculated results, we propose a novel potent inhibitor having a large binding affinity to TLN.

Specific interactions and binding free energies between thermolysin and dipeptides: molecular simulations combined with ab initio molecular orbital and classical vibrational analysis.

Thermolysin (TLN) is a metalloprotease widely used as a nonspecific protease for sequencing peptide and synthesizing many useful chemical compounds by the chemical industry. It was experimentally shown that the activity and functions of TLN are inhibited by the binding of many types of amino acid dipeptides. However, the binding mechanisms between TLN and dipeptides have not been clarified at the atomic and electronic levels. In this study, we investigated the binding mechanisms between TLN and four dipeptides. Specific interactions and binding free energies (BFEs) between TLN and the dipeptides were calculated using molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital (FMO) methods. The molecular systems were embedded in solvating water molecules during calculations. The calculated BFEs were qualitatively consistent with the trend of the experimentally observed inhibition of TLN activity by binding of the dipeptides. In addition, the specific interactions between the dipeptides and each amino acid residue of TLN or solvating water molecules were elucidated by the FMO calculations.

Rapid and quantitative disulfide bond formation for a polypeptide chain using a cyclic selenoxide reagent in an aqueous medium.

To elucidate the reaction mechanism of the disulfide (SS) bond formation reaction of a polypeptide molecule with a water-soluble selenoxide reagent, trans-3,4-dihydroxyselenolane oxide (DHS(ox)), short-term oxidation experiments were carried out for the reduced state (R) of a recombinant hirudin CX-397 variant at pH 7.0 and 25 °C. In the reaction, R was oxidized sequentially to one-SS, two-SS, and three-SS intermediate ensembles within 1 min. The kinetic analysis revealed that the three second-order rate constants for the SS formation are proportional to the number of thiol groups existing in the reactant SS intermediates, indicating the stochastic nature of the SS formation. Ab initio calculation at the HF/6-31++G(d,p) level in water by using the polarizable continuum model suggested that the SS formation reaction is highly exothermic and proceeds via a reactive thioselenurane intermediate with a distorted linear O-Se-S linkage. The results clearly demonstrated that the rate-determining step of the SS formation reaction is the first bimolecular process between a thiol substrate and DHS(ox) rather than the subsequent process to release a SS product.

One-electron redox processes in a cyclic selenide and a selenoxide: a pulse radiolysis study.

One-electron redox reactions of cyclic selenium compounds, DL-trans-3,4-dihydroxy-1-selenolane (DHS(red)), and DL-trans-3,4-dihydroxy-1-selenolane oxide (DHS(ox)) were carried out in aqueous solutions using nanosecond pulse radiolysis, and the resultant transients were detected by absorption spectroscopy. Both *OH radical and specific one-electron oxidant, Br(2)(*-) radical reacted with DHS(red) to form similar transients absorbing at 480 nm, which has been identified as a dimer radical cation (DHS(red))(2)(*+). Secondary electron transfer reactions of the (DHS(red))(2)(*+) were studied with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS(2-)) and superoxide (O(2)(*-)) radicals. The bimolecular rate constants for the electron transfer reaction between (DHS(red))(2)(*+) with ABTS(2-) was determined as 2.4 +/- 0.4 x 10(9) M(-1) s(-1). From this reaction, the yield of (DHS(red))(2)(*+) formed on reaction with *OH radical was estimated in the presence of varying phosphate concentrations. (DHS(red))(2)(*+) reacted with O(2)(*-) radical with a bimolecular rate constant of 2.7 +/- 0.1 x 10(9) M(-1) s(-1) at pH 7. From the same reaction, the positive charge on (DHS(red))(2)(*+) was confirmed by the kinetic salt effect. HPLC analysis of the products formed in the reaction of (DHS(red))(2)(*+) with O(2)(*-) radicals showed formation of the selenoxide, DHS(ox). In order to know if a similar mechanism operated during the reduction of DHS(ox), its reactions with e(aq)(-) were studied at pH 7. The rate constant for this reaction was determined as 5.6 +/- 0.9 x 10(9) M(-1) s(-1), and no transient absorption could be observed in the wavelength region from 280 to 700 nm. It is proposed that the radical anion (DHS(ox))(*-) formed by a one-electron reduction would get protonated to form a hydroxyl radical adduct, which in presence of proton donors, would undergo dehydration to form DHS(*+). Evidence for this mechanism was obtained by converting DHS(*+) to (DHS(red))(2)(*+) with the addition of DHS(red) to the same system. Quantum chemical calculations provided supporting evidence for some of the redox reactions.

Specific interactions between aryl hydrocarbon receptor and dioxin congeners: ab initio fragment molecular orbital calculations.

Aryl hydrocarbon receptor (AhR) is a transcription factor and its function is activated by the binding of halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,4-trichlorodibenzo-p-dioxin (TrCDD). TCDD is highly toxic to rat, whereas its congener TrCDD shows only a weak effect on gene expression. In order to elucidate the reason of this remarkable difference in the effect of TCDD and TrCDD, we here obtained stable structures of the complexes with rat AhR (rAhR) and TCDD/TrCDD and investigated their electronic properties by using the ab initio fragment molecular orbital (FMO) method. The results indicate that TCDD binds more strongly to rAhR than TrCDD, which is consistent with the experimentally observed toxicity of TCDD and TrCDD. Furthermore, ab initio FMO calculations elucidate that His324 and Gln381 of rAhR are important for binding TCDD, while His324 and Ser334 are important for TrCDD binding.

Hybridization energies of double strands composed of DNA, RNA, PNA and LNA.

The electronic properties of double strands composed of trimeric LNA, PNA, DNA and RNA single strands were investigated by density-functional molecular orbital calculations. The computed hybridization energies for the double strands involving PNA or LNA are larger than those for DNA-DNA and RNA-RNA. The larger stability is attributed to the presence of a larger positive charge of the hydrogen atoms contributing to the hydrogen bonds in the PNA-DNA and LNA-DNA double-strands. These results are comparable to the experimental finding that PNA and LNA single strands display high affinity toward a complementary DNA or RNA single strand.

A combined molecular dynamics/density-functional theoretical study on the structure and electronic properties of hydrating water molecules in the minor groove of decameric DNA duplex.

A combined molecular dynamics/density-functional theoretical study was carried out to address the propensity of ambient water to form cross-strand bridging water (CSBW) and their effects on the electronic properties of a fully hydrated DNA duplex 5'-d(CCATTAATGG)(2)-3'. The simulation shows ubiquitous presence of up to five CSBWs along the minor groove, each with residence time ranging from 400 ps to 750 ps. The molecular orbitals localized on these CSBWs are nearly degenerate in energy with the highest occupied molecular orbital of DNA localized on guanine bases, strongly indicating that the hole transport along the guanines is mediated by the ubiquitous CSBWs.

Charge Transfer Through Single- and Double-strand DNAs: Simulations Based on Molecular Dynamics and Molecular Orbital Methods.

To elucidate the difference in charge transfer through single- and double-strand DNAs, we performed simulations based on classical molecular dynamics (MD) and semiempirical molecular orbital (MO) methods. Stable structures of DNA strands in water were determined by MD simulations and their energy levels and distributions of frontier MOs that mediate charge transfer were analyzed by the MO calculations. The transfer integrals for a hole or an electron between the neighboring DNA bases were estimated from the energy levels of frontier MOs. We obtained the current-voltage characteristics for single- and double-strand DNAs that are qualitatively in agreement with the experimental results.

Effect of base mismatch on the electronic properties of DNA-DNA and LNA-DNA double strands: Density-functional theoretical calculations.

The electronic properties of double-stranded octametric DNA-DNA and LNA-DNA with a single-base mismatch were compared with those having fully complementary base pairs to quantify the effect of the base mismatch on hybridization energies (HE). A single T-G mismatch in the LNA-DNA gives rise to a significant reduction in HE, which is consistent with a significant lowering of the melting temperature for mismatched LNA-DNA. By contrast, the hybridization strength of the mismatched DNA-DNA depends strongly on local hydrogen-bonding arrangements in the mispaired T-G. The difference in HE is explained in terms of variation in charge distributions around the hydrogen-bonded base pairs.