Abstract

Here, we present an update of the CHARMM27 all-atom additive force field for nucleic acids that improves the treatment of RNA molecules. The original CHARMM27 force field parameters exhibit enhanced Watson-Crick base pair opening which is not consistent with experiment, whereas analysis of molecular dynamics (MD) simulations show the 2'-hydroxyl moiety to almost exclusively sample the O3' orientation. Quantum mechanical (QM) studies of RNA related model compounds indicate the energy minimum associated with the O3' orientation to be too favorable, consistent with the MD results. Optimization of the dihedral parameters dictating the energy of the 2'-hydroxyl proton targeting the QM data yielded several parameter sets, which sample both the base and O3' orientations of the 2'-hydroxyl to varying degrees. Selection of the final dihedral parameters was based on reproduction of hydration behavior as related to a survey of crystallographic data and better agreement with experimental NMR Jcoupling values. Application of the model, designated CHARMM36, to a collection of canonical and noncanonical RNA molecules reveals overall improved agreement with a range of experimental observables as compared to CHARMM27. The results also indicate the sensitivity of the conformational heterogeneity of RNA to the orientation of the 2'-hydroxyl moiety and support a model whereby the 2'- hydroxyl can enhance the probability of conformational transitions in RNA.

Abstract

Background: The extracellular signal-regulated kinase-1 and 2 (ERK1/2) proteins play an important role in cancer cell proliferation and survival. ERK1/2 proteins also are important for normal cell functions. Thus, anti-cancer therapies that block all ERK1/2 signaling may result in undesirable toxicity to normal cells. As an alternative, we have used computational and biological approaches to identify low-molecular weight compounds that have the potential to interact with unique ERK1/2 docking sites and selectively inhibit interactions with substrates involved in promoting cell proliferation. Methods: Colony formation and water soluble tetrazolium salt (WST) assays were used to determine the effects of test compounds on cell proliferation. Changes in phosphorylation and protein expression in response to test compound treatment were examined by immunoblotting and in vitro kinase assays. Apoptosis was determined with immunoblotting and caspase activity assays. Results: In silico modeling was used to identify compounds that were structurally similar to a previously identified parent compound, called 76. From this screen, several compounds, termed 76.2, 76.3, and 76.4 sharing a common thiazolidinedione core with an aminoethyl side group, inhibited proliferation and induced apoptosis of HeLa cells. However, the active compounds were less effective in inhibiting proliferation or inducing apoptosis in non-transformed epithelial cells. Induction of HeLa cell apoptosis appeared to be through intrinsic mechanisms involving caspase-9 activation and decreased phosphorylation of the pro-apoptotic Bad protein. Cell-based and in vitro kinase assays indicated that compounds 76.3 and 76.4 directly inhibited ERK-mediated phosphorylation of caspase-9 and the p90Rsk-1 kinase, which phosphorylates and inhibits Bad, more effectively than the parent compound 76. Further examination of the test compound’s mechanism of action showed little effects on related MAP kinases or other cell survival proteins. Conclusion: These findings support the identification of a class of ERK-targeted molecules that can induce apoptosis in transformed cells by inhibiting ERK-mediated phosphorylation and inactivation of pro-apoptoticproteins.

Abstract

Transannular Diels Alder reaction (TADA), a combination of both intramolecular and Diels-Alder reactions, is a powerful synthetic organic reaction mechanism used in synthesizing polycyclic compounds with high degree of chemo-, regio- and stereospecificity. TADA reactions occur in (x+y+2)-membered triene macrocycles, which contain both the diene and the dienophile moieties, to form A.B.C[x+6+y] type tricyclic compounds. The TADA reactivity of a series of 14-membered macrocyclic rings have been studied using the density functional B3LYP level of theory. The reactants and the transition states are capable of existing in different conformations. The key conformers of the transition states were studied at the B3LYP level. We have done force field parameters for the trienes, and performed detailed replica exchange molecular dynamics simulations to identify all possible isomers of the reactants. We established a linear correlation between the activation energies and the extent of sampling of the reactants in certain conformational states. In this presentation, we also will present the effect of the lengths of the linkers (3 to 5 methylene units) connecting the diene and dienophile units on the TADA reactivities.

Abstract

Ribonucleases act as biological counter weights by playing a major role in gene expression. Ribonuclease H is one of them which performs the endonucleotic cleaveage reaction of the 3'-O-P linkage of RNA strand of DNA/RNA hybrids. These enzymes show specificity towards DNA/RNA hybrids but not pure structures. One of the reason for this differentiation could be mixed A/Bconformation of hybrid compared to pure structures and there might be other reasons. There is a debate concerning the substrate recognition mechanism whether this is because of induced fit type mechanism or conformational rearrangement of monomers in order to come close or may be non bonded interactions which will put the enzyme and substrate together in order to perform the hydrolysis reaction. While the role of metal ions in the reaction has been recognised, the mechanism is not obvious. For differentating the structural changes, we performed long molecular dynamics (MD) simulations on ribonuclease H enzyme of B.halodurans with its substrate and their monomers. The calculations show that the large conformational changes in both the monomers favours the reaction. Since the protein hydrophobic core is very stable, these changes will take place at surface level. The principal component analysis also supports the large conformational changes in protein and hybrid. Apart form these changes, the protein binding increases the plasticity of the hybrids. Overall the calculations show that structural deformations in both hybrid and enzyme brings them close to each other. Free energy calculations have been performed to study the proteinhybrid interactions

Abstract

Understanding protein folding thermodynamics and kinetics is a central issue in molecular biology. Molecular dynamics (MD) simulations are being extensively used for this purpose. However, direct comparison between simulations and experiments requires both, an accurate description of the system and extensive sampling of the conformational space. Variants of MD have been developed to overcome sampling issues. Here, we use umbrella sampling technique to quantitatively compute the differences in thermodynamic quantities of the unfolding process of Trp-cage miniprotein TC5b (PDB ID: 1L2Y) due to changes in temperature as well as the presence/absence of chemical denaturants. Urea at a concentration of 8M has been used as the chemical denaturant. WHAM was used to obtain the unbiased free energy profiles. The free energy profile can be categorised into three parts – the opening of the Trp-cage, separation of secondary structure elements and distortion of alpha-helix. An increase in temperature or presence of urea effects only the opening of the Trp-cage while the separation of the secondary structure elements and distortion of alpha-helix is independent of temperature and urea. Contour plots were constructed to substantiate the results obtained from free energy profiles.

Abstract

Identification of the structural and energetic determinants responsible for enhancing the stability of proteins is crucial. Hyperthermophilic proteins are naturally occurring proteins that exhibit high thermal stability and are good candidates for the investigation and understanding of structure-stability relationships. Sac7d from Sulfolobus acidocaldarius and Sso7d from Sulfolobus solfactaricus are two homologous hyperthermophilic proteins that were shown to be quite stable at high temperatures. Molecular dynamics simulations at the nanosecond time scale at different temperatures were performed to examine the factors affecting their stability. The three-dimensional structures of these proteins were observed to be similar to the experimental structure at 300 and 360 K but were found to undergo denaturation at 500 K. Both proteins exhibit similar unfolding pathways that correlates well with the calculated intermolecular interaction energies. The differential dynamic behaviors of these molecules at different temperatures were examined. Structural and energetic analysis of the contributions of salt bridges indicates a stabilizing effect at higher temperatures. However, the lifetimes of the salt bridges were found to be quite short, and several new salt bridges formed at 500 K supporting previous studies that the desolvation penalty due to the formation of salt bridges decreases at elevated temperatures. Hydrophobic interactions, which decrease with increase in temperature, were also found to be crucial in the stability of these proteins. Overall, the study shows that a balance among the salt bridge interactions, hydrophobic interactions, and solvent properties is primarily responsible for the high thermal stability of this class of proteins.

Abstract

SAM-III riboswitch, involved in regulating sulfur metabolic pathways in lactic acid bacteria, is capable of differentiating S-adenosyl-L-methionine (SAM) from its structurally similar analog S-adenosyl-L-homocysteine (SAH). Atomic level understanding of the ligand recognition mechanism of riboswitches is essential for understanding their structure-function relationships in general. In the present study, we have employed molecular dynamics (MD) simulations on five model systems to elucidate the discrimination mechanism adopted by the SAM-III riboswitch that enables differential binding of SAM with respect to SAH. The structures of the binding pocket of the riboswitch, and the modes of binding of the adenine moiety of SAM obtained from the MD simulations are similar to the experimental structure. However, MD simulations of the riboswitch-SAH complexes lead to partial unbinding of the ligand and structural changes in the RNA binding pocket. Detailed analyses were performed to examine the structural and energetic factors involved in such a differentiation. The calculations reveal a novel mechanism by which the aptamer domain specifically recognizes the adenine moiety of SAM/SAH, but SAM is better stabilized in the binding pocket due to nonspecific electrostatic interactions involving the sulfonium group. Additionally, the results support less dependence of the ligand conformation in the bound form on the effective binding of SAM to the riboswitch.

Abstract

DNA-RNA hybrids are heterogeneous nucleic acid duplexes that contain a DNA strand and a RNA strand. They are identified as key intermediates in transcription, and in other biological processes. These novel molecules have also been recognized as potential candidates in antisense therapy. While DNA exists in B-form, and RNA in A-form, the process of hybridization between these two distinct conformations, and the resultant structural features of the hybrid duplexes are interesting in their own right. It has been suggested that hybrids have unique conformational properties that form the basis of Ribonuclease H (RNase H) activity only on these molecules, and not on pure DNA or RNA. Quantitative examination of the conformational properties of hybrids is very important for understanding the RNase H activity. Several nanosecond long molecular dynamics (MD) simulations were performed on all the available structures in presence of explicit solvent environment. All the MD simulations were performed using the CHARMM27 all atom nucleic acid force field. In addition to the MD simulations on hybrids, control simulations were also performed on pure DNA and RNA duplexes with identical sequences. Several structural, dynamic and energetic properties including deviations, flexibility, intramolecular entropy, helical properties, and backbone conformational preferences were computed. The conformational properties of the sugar puckering angles revealed interesting trends that depend on the sequence of the duplex. RNA strands in all the duplexes sample conformational regions corresponding to a pure-A form nucleic acid. However, the DNA strands adopt mixing of A- and B- type conformations resulting in intermediate structures for the hybrids. The extent of this mixing depends primarily on the relative purine to pyrimidine composition ratio in the DNA strands, and sequence effects. The presentation will focus on the structural and dynamic properties of hybrid duplexes in comparison with those of pure-RNA and DNA duplexes.

Abstract

Riboswitches are RNA-based genetic control elements that function via a conformational transition mechanism when a specific target molecule binds to its binding pocket. To facilitate an atomic detail interpretation of experimental investigations on the role of the adenine ligand on the conformational properties and kinetics of folding of the add adenine riboswitch, we performed molecular dynamics simulations in both the presence and the absence of the ligand. In the absence of ligand, structural deviations were observed in the J23 junction and the P1 stem. Destabilization of the P1 stem in the absence of ligand involves the loss of direct stabilizing interactions with the ligand, with additional contributions from the J23 junction region. The J23 junction of the riboswitch is found to be more flexible, and the tertiary contacts among the junction regions are altered in the absence of the adenine ligand; results suggest that the adenine ligand associates and dissociates from the riboswitch in the vicinity of J23. Good agreement was obtained with the experimental data with the results indicating dynamic behavior of the adenine ligand on the nanosecond time scale to be associated with the dynamic behavior of hydrogen bonding with the riboswitch. Results also predict that direct interactions of the adenine ligand with U74 of the riboswitch are not essential for stable binding although it is crucial for its recognition. The possibility of methodological artifacts and force-field inaccuracies impacting the present observations was checked by additional molecular dynamics simulations in the presence of 2,6-diaminopurine and in the crystal environment. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Abstract

Sac7d belongs to a family of chromosomal proteins, which are crucial for thermal stabilization of DNA at higher growth temperatures. It is capable of binding DNA nonspecifically, and is responsible for the increase in the melting temperature of DNA in the bound form up to 85 °C. Molecular dynamics (MD) simulations were performed at different temperatures on two protein-DNA complexes of Sac7d. Various structural and energetic parameters were calculated to examine the DNA stability and to investigate the conformational changes in DNA and the protein-DNA interactions. Room temperature simulations indicated very good agreement with the experimental structures. The protein structure is nearly unchanged at both 300 and 360 K, and only up to five base pairs of the DNA are stabilized by Sac7d at 360 K. However, the MD simulations on DNA alone systems show that they lose their helical structures at 360 K further supporting the role of Sac7d in stabilizing the oligomers. At higher temperatures (420 and 480 K), DNA undergoes denaturation in the presence and the absence of the protein. The DNA molecules were found to undergo B- to A-form transitions consistent with experimental studies, and the extent of these transitions are examined in detail. The extent of sampling B- and A-form regions was found to show temperature and sequence dependence. Multiple MD simulations yielded similar results validating the proposed model. Interaction energy calculations corresponding to protein-DNA binding indicates major contribution due to DNA backbone, explaining the nonspecific interactions of Sac7d.