Abstract

Schistosomiasis is a disease caused by a trematode parasite, Schistosoma mansoni. The host consists of two different redox systems: Glutaredoxin (Grx) and Thioredoxin (Trx), but the parasite has only one multifunctional enzyme Thioredoxin Glutathione Reductase (TGR), which is responsible for carrying out all the redox activities, necessary for protecting the parasite against oxidative stress inside the host. TGR is a fusion of Thioredoxin Reductase (TR) with Glutaredoxin (Grx) at its N-terminus. The dependence of S. mansoni on a single protein for all its redox functions makes it a potential drug target. We have carried out structural analysis of Glutathione Reductase (GR), Thioredoxin Reductase (TR) and Thioredoxin Glutathione Reductase (TGR) using available crystal structures from PDB to understand the catalytic mechanism in these oxidoreductases. NADPH plays a major role as an electron carrier and transfers the electrons to the oxidized cofactor, FAD, which is non-covalently attached to the enzyme. This initiates further steps of the catalytic process. Here, we investigate how the interplay of conformations of these cofactors and those of the neighbouring residues facilitate the charge transfer process. TR, GR and TGR share similarity in their respective active site regions. Several conserved residues and non covalent interactions, play a major role during the electron transfer process. We hypothesise the function of NADPH during the entire charge transfer process as follows: a Tyr residue moves and facilitates the binding of NADPH to FAD in pi-stacking conformation. Two Arg residues reorient to interact with 2'-phosphate group and adenine ring of NADPH for effective binding. Lys, Glu and His residues near the active site regions form salt bridges and possibly aid the charge transfer mechanism in these redox enzymes.

Abstract

BACKGROUND: Thermostable lipases are widely used in the detergent industry and in organic synthesis. B. subtilis lipase (Lip A) is a monomeric protein, which preferentially hydrolyses C8 fatty acid esters. Though the mechanism of thermostability depends on type of lipase, some common factors include additional non-covalent interactions, increase in packing density of the hydrophobic core, stabilization of alpha-helices and shortening of loops between secondary structural elements. RESULTS: We have studied wild type Lip A (WT) and six of its thermostable mutants containing 2, 3, 4, 6, 9 and 12 point mutations respectively, derived from the wild type protein by directed evolution. Experimental observations showed that the most thermostable variant, with twelve mutations, shows remarkable enhancement in melting temperature and temperature optima of activity compared to WT, in vitro. Our MD simulations show decrease in SASA for core regions and slow unfolding rate at several key secondary structural regions due to presence of persistent salt bridges, hydrogen bonds and native contacts for the most thermostable mutant lipase. The principal component (PC) and Free energy landscape (FEL) analyses at 450K suggest that the most thermostable mutant showed least fluctuation and stayed within a single large minimum. The other less thermostable mutants and WT showed higher fluctuation and exhibited two or more minima along the first two PCs. Analysis of representative structures corresponding to each sub-states showed that the most thermostable mutant sampled conformations close to the native state most of the time during the simulation. The other less thermostable mutants and WT lipase sampled non-native conformations more frequently than native conformations. CONCLUSIONS: Our results demonstrate that, the most thermostable lipase maintains its stability and activity at high temperatures by restricting conformational fluctuation through the stabilization of key salt bridges and main-chain H-bonds, which further helped them to maintain the stability of the core regions apart from stabilizing the overall 3D fold.

Abstract

Archaeal chromatin proteins share molecular and functional similarities with both bacterial and eukaryotic chromatin proteins. These proteins play an important role in functionally organizing the genomic DNA into a compact nucleoid. Cren7 and Sul7 are two crenarchaeal nucleoidassociated proteins, which are structurally homologous, but not conserved at the sequence level. Cocrystal structures have shown that these two proteins induce a sharp bend on binding to DNA. In this study, we have investigated the architectural properties of these proteins using atomic force microscopy, molecular dynamics simulations and magnetic tweezers. We demonstrate that Cren7 and Sul7 both compact DNA molecules to a similar extent. Using a theoretical model, we quantify the number of individual proteins bound to the DNA as a function of protein concentration and show that forces up to 3.5 pN do not affect this binding. Moreover, we investigate the flexibility of the bending angle induced by Cren7 and Sul7 and show that the protein–DNA complexes differ in flexibility from analogous bacterial and eukaryotic DNA-bending proteins.

Abstract

The effect of the linker length (–CH2–) connecting the diene and the dienophile in macrocyclic trienes on their transannular Diels–Alder (TADA) reactivity has been investigated using density functional theory (DFT) calculations. The relationship between the conformational properties of these reactants and their reaction energy barriers was examined and a quantitative relationship has been obtained. The transition state energy barriers were found to increase with an increase in the linker length, which is in contrast to the expected trend. The conformational preferences of the triene reactants were studied using replica exchange molecular dynamics (REMD) simulations. These calculations reveal that longer linkers lead to a decreased probability of the occurrence of conformations with the diene and dienophile parts of the system vicinal to each other, and thus lower reactivity of systems with long linkers. Excellent correlations between the transition state energies and the cumulative probabilities corresponding to a short distance between the reactive sites, along with the ability of the diene to exist in the s-cisoid form, were observed.

Abstract

Locked nucleic acid (LNA) is a chemical modification which introduces a -O-CH2-linkage in the furanose sugar of nucleic acids and blocks its conformation in a particular state. Two types of modifications, namely, 2'-O,4'-C-methylene-β-D-ribofuranose (β-D-LNA) and 2'-O,4'-C-methylene-α-L-ribofuranose (α-L-LNA), have been shown to yield RNA and DNA duplex-like structures, respectively. LNA modifications lead to increased melting temperatures of DNA and RNA duplexes, and have been suggested as potential therapeutic agents in antisense therapy. In this study, molecular dynamics (MD) simulations were performed on fully modified LNA duplexes and pure DNA and RNA duplexes sharing a similar sequence to investigate their structure, stabilities, and solvation properties. Both LNA duplexes undergo unwinding of the helical structure compared to the pure DNA and RNA duplexes. Though the α-LNA substituent has been proposed to mimic deoxyribose sugar in its conformational properties, the fully modified duplex was found to exhibit unique structural and dynamic properties with respect to the other three nucleic acid structures. Free energy calculations accurately capture the enhanced stabilization of the LNA duplex structures compared to DNA and RNA molecules as observed in experiments. π-stacking interaction between bases from complementary strands is shown to be one of the contributors to enhanced stabilization upon LNA substitution. A combination of two factors, namely, nature of the -O-CH2- linkage in the LNAs vs their absence in the pure duplexes and similar conformations of the sugar rings in DNA and α-LNA vs the other two, is suggested to contribute to the stark differences among the four duplexes studied here in terms of their structural, dynamic, and energetic properties

Abstract

The emergence of single-molecule force measurement experiments has facilitated a better understanding of protein folding pathways and the thermodynamics involved. Computational methods such as steered molecular dynamics (SMD) simulations are helpful in providing atomistic level information on the unfolding pathways. Recent experimental studies have showed that combinations of single-molecule experiments with traditional methods such as chemical and/or thermal denaturation yield additional insights into the folding phenomenon. In this study, we report results from extensive computations (a total of about 60 SMD simulations with a total length of about 0.4 μs) that address the effect of thermal perturbation on the mechanical stability of the I27 domain of the protein titin. A wide range of temperatures (280–340 K) were considered for the pulling, which was done at both constant velocity and constant force using SMD simulations. Good agreement with experimental data, such as for the trends in changes in average force and the maximum force with respect to the temperature, was obtained. This study identifies two competing pathways for the mechanical unfolding of I27, and illustrates the significance of combining various techniques to examine protein foldin

Abstract

The role of salt bridges in chromatin protein Sso7d, from S. solfataricus has previously been shown to be crucial for its unusual high thermal stability. Experimental studies have shown that single site mutation of Sso7d (F31A) leads to a substantial decrease in the thermal stability of this protein due to distortion of the hydrophobic core. In the present study, we have performed a total of 0.2s long molecular dynamics (MD) simulations on F31A at room temperature, and at 360 K, close to the melting temperature of the wild type (WT) protein to investigate the role of hydrophobic core on protein stability. Sso7d-WT was shown to be stable at both 300 and 360 K; however, F31A undergoes denaturation at 360 K, consistent with experimental results. The structural and energetic properties obtained using the analysis of MD trajectories indicate that the single mutation results in high flexibility of the protein, and loosening of intramolecular interactions. Correlation between the dynamics of the salt bridges with the structural transitions and the unfolding pathway indicate the importance of both salt bridges and hydrophobic in effecting thermal stability of proteins in general.

Abstract

A series of dihydrogen complexes trans-[Ru(η2-H2){SC(SR)H}(dppe)2][X][BF4] (R = CH3, X = OTf; R = C6H5CH2, X = BPh4; R = H2C=CHCH2, X = BPh4; dppe = Ph2PCH2CH2PPh2) bearing sulfur-donor ligands has been synthesized by protonation of the (alkyl dithioformate)hydrido complexes trans-[Ru(H){SC(SR)H}(dppe)2][X] by using HBF4•Et2O. Competitive substitution reactions between H2 and SC(SR)H in trans-[Ru(η2-H2){SC(SR)H}(dppe)2][X][BF4] have been studied by treatment with CH3CN, CO, and P(OCH3)3. These resulted in the expulsion of SC(SR)H from the metal center, thus indicating that the alkyl dithioformate ligand is more labile than H2. Bonding of alkyl dithioformate ligands (sulfur-donor ligands) trans to H2 have been studied by comparing the H–H distances and chemical-shift values (1H NMR spectroscopy) of the various dihydrogen complexes bearing different trans ligands. This study qualitatively suggests that the alkyl dithioformate ligands in these trans-dihydrogen complexes show a poor π effect, and it is further supported by density functional theory calculations. The first example of a dihydrogen complex bearing dithioformic acid, trans-[Ru(η2-H2){SC(SH)H}(dppe)2][BF4]2, was obtained by protonation of trans-[Ru(H){SC(S)H}(dppe)2] by using HBF4•Et2O.

Abstract

In the late 19th century, van’t Hoff and Le Bel proposed that carbon prefers a tetrahedral arrangement in its tetracoordinate form. Similarly, the isoelectronic species such as B-, N+, Al-, Si and P+ assume tetrahedral arrangement. However, it has been shown by several research groups that planar arrangement for tetracoordinated carbon centers may be possible for certain molecules that are stabilized by steric constraints and electronic effects. Sastry and coworkers have identified a series of neutral hydrocarbons that are stable in their planar form. We have done high level quantum chemical calculations on skeletally substituted derivative of one of the hydrocarbons (C5H4). Interestingly, the phosphonium ion (C4PH4 +) is found to be a minimum on its potential energy surface consistently by both density functional theory and MP2 methods. A transition state corresponding to the interconversion between the two forms was identified, which indicates rapid transitions. Additionally, the tetrahedral form was found to be the minima for C and B-. The tetrahedral form is the minima for Al- and Si, whereas the planar form was characterized as a transition state that connects two identical tetrahedral forms. Ab initio molecular dynamics calculations indicate that C4H4Si does undergo rapid interconversion between two identical tetrahedral structures through the planar transition state in the femtosecond timescale. However, C4H4P+ and C4H4Al- undergoes irreversible ring opening during the simulations. These molecules/ions were further analyzed using NICS calculations and ring opening reactivities.

Abstract

Due to poor stability and nature of low nuclease resistance of pure nucleic acids, a suitable chemical modification is required which improves several factors such as increased cellular uptake, altered duplex conformation and stability. In general, the modifications at ribose sugar (backbone) of the oligonucleotide attribute special features to the duplexes than other modifications. Some of the common modifications include 2'-O-methyl, 2'-O,4'-C-methylene-β-D-ribofuranosyl (LNA) and phosphoroamidate. The modified oligonucleotides have also been shown to be potential antisense agents. Molecular dynamics (MD) simulations are valuable tools to study nucleic acid dynamics, conformational preferences, stabilities etc. Here, we have employed different simulation techniques such as traditional MD, replica exchange MD to understand the properties of modified duplexes and also the origin for the effect of chemical modification. The simulations revealed several important aspects related to duplex stability and relation with their base content. The factors responsible for the stability/unstability of modified duplexes with different sequences are also examined. Our results also suggest that the differential changes observed in nucleic acids depends on the position, nature of the modified site. This study also provides a molecular level picture of structural changes arising due to different types of modifications which is important in designing antisense oligonucleotides.