Microsecond Scale Replica Exchange Molecular Dynamic Simulation of Villin Headpiece: An Insight into the Folding Landscape

Abstract

Reaching the experimental time scale of millisecond is a grand challenge for protein folding simulations. The development of advanced Molecular Dynamics techniques like Replica Exchange Molecular Dynamics (REMD) makes it possible to reach these experimental timescales. In this study, an attempt has been made to reach the multi microsecond simulation time scale by carrying out folding simulations on a three helix bundle protein, Villin, by combining REMD and Amber United Atom model. Twenty replicas having different temperatures ranging from 295 K to 390 K were simulated for 1.5 μs each. The lowest Root Mean Square Deviation (RMSD) structure of 2.5 Å was obtained with respect to native structure (PDB code 1VII), with all the helices formed. The folding population landscapes were built using segment-wise RMSD and Principal Components as reaction coordinates. These analyses suggest the two-stage folding for Villin. The combination of REMD and Amber United Atom model may be useful to understand the folding mechanism of various fast folding proteins     

Hypoglycemic activity of alkaloidal fraction of Tinospora cordifolia

The stem of Tinospora cordifolia (TC) is widely used in the therapy of diabetes in traditional folk medicine of India. In the present study, isoquinoline alkaloid rich fraction (AFTC) derived from stem of TC and three alkaloids viz., palmatine, jatrorrhizine and magnoflorine were evaluated for insulin-mimicking and insulin-releasing effect in vitro and in vivo. Their effect on hepatic gluconeogenesis was examined in rat hepatocytes. Insulin releasing effect was detected in vitro using rat pancreatic β-cell line, RINm5F. Furthermore, effects of AFTC and isolated alkaloids on serum glucose and insulin level were studied in fasted and glucose challenged normal rats. AFTC significantly decreased gluconeogenesis in rat hepatocytes as insulin did and it increases insulin secretion in RINm5F cells similar to tolbutamide. In acute 30 min test in vitro, AFTC, palmatine, jatrorrhizine and magnoflorine stimulated insulin secretion from the RINm5F cell line. As in vivo results, administration of AFTC (50, 100, and 200 mg/kg), palmatine, jatrorrhizine and magnoflorine (10, 20 and 40 mg/kg each) orally significantly decreased fasting serum glucose, and suppressed the increase of blood glucose levels after 2 g/kg glucose loading in normal rats. In vivo study further justified their insulin secreting potential by raising the serum insulin level in glucose fed rats. These results demonstrate the alkaloid present in TC contributed for antihyperglycemic activity. AFTC may have hypoglycemic effects via mechanisms of insulin releasing and insulin-mimicking activity and thus improves postprandial hyperglycemia.     

Cavities and Atomic Packing in Protein Structures and Interfaces

A comparative analysis of cavities enclosed in a tertiary structure of proteins and interfaces formed by the interaction of two protein subunits in obligate and non-obligate categories (represented by homodimeric molecules and heterocomplexes, respectively) is presented. The total volume of cavities increases with the size of the protein (or the interface), though the exact relationship may vary in different cases. Likewise, for individual cavities also there is quantitative dependence of the volume on the number of atoms (or residues) lining the cavity. The larger cavities tend to be less spherical, solvated, and the interfaces are enriched in these. On average 15 ų of cavity volume is found to accommodate single water, with another 40–45 ų needed for each additional solvent molecule. Polar atoms/residues have a higher propensity to line solvated cavities. Relative to the frequency of occurrence in the whole structure (or interface), residues in β-strands are found more often lining the cavities, and those in turn and loop the least. Any depression in one chain not complemented by a protrusion in the other results in a cavity in the protein–protein interface. Through the use of the Voronoi volume, the packing of residues involved in protein–protein interaction has been compared to that in the protein interior. For a comparable number of atoms the interface has about twice the number of cavities relative to the tertiary structure.     

Interaction between supernodulating or non-nodulating mutants of soybean and two arbuscular mycorrhizal fungi

 Twelve nodulation mutants (seven non-nodulating and five supernodulating) of soybean [Glycine max (L.) Mirr.] were screened for arbuscular mycorrhizal colonization in the presence of either Glomus etunicatum Becker and Gerdemann or Gigaspora margarita Becker and Hall. The cultivars showed variation in colonization parameters. The two supernodulating mutants En6500 and NOD1–3 had higher frequencies of colonization with 2.5–4.5 times higher arbuscular abundance than the respective wild types. The enhanced mycorrhization resulted in significant enhancement of P uptake by En6500. The non-nodulating mutants showed decreases in mycorrhizal parameters. Mutants En1282 and Harosoy^–exhibited aborted infection after formation of typical appressorium-like structures at some sites. However, none of these had the non-mycorrhizal phenotype. Growth and nutrient-uptake parameters should be considered while studying plant mutants for mycorrhization. Accepted: 7 July 2000     

Conformational preferences of anticodon 3'-adjacent hypermodified nucleic acid base cis-or trans-zeatin and its 2-methylthio derivative, cis-or trans- ms(2)zeatin

Conformational preferences of the hypermodified nucleic acid bases N6-(Delta(2)-cis-hydroxyisopentenyl)adenine, cis-io(6)Ade also known as cis-zeatin, and N(6)-(Delta(2)-trans-hydroxyisopentenyl)adenine, trans-io(6)ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms(2)io(6)Ade or cis-ms(2)zeatin, and trans-ms(2)io6Ade or trans-ms(2)zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AM1 and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io(6)Ade, trans-io(6)Ade, cis-ms(2)io(6)Ade and trans-ms(2)io(6)Ade are such that in each of these molecules the isopentenyl substituent spreads away (has "dista" conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io(6)Ade as well as cis-ms(2)io(6)Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io(6)Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms(2)io(6)Ade, possible O-H...S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms(2)io(6)Ade as well as trans-ms(2)io(6)Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this non-obstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i(6)Ade and ms(2)i(6)Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms(2)-zeatin (ms(2)io(6)Ade) modification.     

Dissolution of sulphur particles by Thiobacillus ferrooxidans: substrate for unattached cells

The growth of Thiobacillus ferrooxidans on sulphur is known to proceed through the attachment of cells to the sulphur particles. Experiments, However, show that the cells in the liquid phase, which are not attached to the sulphur particles, also grow. It has been shown through the use of a two-compartment membrane reactor that this increase is partially due to the release of ions, corresponding to partially oxidized of sulphur, into the solution by the attached cells. The main soluble ion has been found to the thiosulphate, but traces of sulphite have also been detected. (c) 1993 John Wiley & Sons, Inc.     

Study of early events in the protein folding of villin headpiece using molecular dynamics simulation

Protein folding is scientifically and computationally challenging problem. The early phases of protein folding are interesting due to various events like nascent secondary structure formation, hydrophobic collapse leading to formation of non-native or meta-stable conformations. These events occur within a very short time span of 100ns as compared to total folding time of few microseconds. It is highly difficult to observe these events experimentally due to very short lifetime. Molecular dynamics simulation technique can efficiently probe the detailed atomic level understanding about these events. In the present paper, all atom molecular dynamics simulation trajectory of nearly 200ns was carried out for fully solvated villin headpiece with PME treatment using AMBER 7 package. Initial hydrophobic collapse along with secondary structure formation resulted into formation of partially stable non-native conformations. The formation of secondary structural elements and hydrophobic collapse takes place simultaneously in the folding process.     

Microsecond scale replica exchange molecular dynamic simulation of villin headpiece: an insight into the folding landscape

Reaching the experimental time scale of millisecond is a grand challenge for protein folding simulations. The development of advanced Molecular Dynamics techniques like Replica Exchange Molecular Dynamics (REMD) makes it possible to reach these experimental timescales. In this study, an attempt has been made to reach the multi microsecond simulation time scale by carrying out folding simulations on a three helix bundle protein, Villin, by combining REMD and Amber United Atom model. Twenty replicas having different temperatures ranging from 295 K to 390 K were simulated for 1.5 μs each. The lowest Root Mean Square Deviation (RMSD) structure of 2.5 ? was obtained with respect to native structure (PDB code 1VII), with all the helices formed. The folding population landscapes were built using segment-wise RMSD and Principal Components as reaction coordinates. These analyses suggest the two-stage folding for Villin. The combination of REMD and Amber United Atom model may be useful to understand the folding mechanism of various fast folding proteins.     

Probing the wild-type HRas activation mechanism using steered molecular dynamics,understanding the energy barrier and role of water in the activation

Ras is one of the most common oncogenes in human cancers. It belongs to a family of GTPases that functions as binary conformational switches by timely switching of their conformations from GDP to GTP and vice versa. It attains the final active state structure via an intermediate GTP-bound state. The transition between these states is a millisecond-time-scale event. This makes studying this mechanism beyond the scope of classical molecular dynamics. In the present study, we describe the activation pathway of the HRas protein complex along the distance-based reaction coordinate using steered molecular dynamics. Approximately ~720 ns of MD simulations using CMD and SMD was performed. We demonstrated the change in orientation and arrangement of the two switch regions and the role of various hydrogen bonds during the activation process. The weighted histogram analysis method was also performed, and the potential of mean force was calculated between the inactive and active via the intermediate state (state 1) of HRas. The study indicates that water seems to play a crucial role in the activation process and to transfer the HRas protein from its intermediate state to the fully active state. The implications of our study hereby suggest that the HRas activation mechanism is a multistep process. It starts from the inactive state to an intermediate state 1 followed by trapping of water molecules and flipping of the Thr35 residue to form a fully active state (state 2). This state 2 also comprises Gly60, Thr35, GTP, Mg²⁺ and water-forming stable interactions.     

Molecular dynamics simulation of drug uptake by polymer

Drug uptake by polymer was modeled using a molecular dynamics (MD) simulation technique. Three drugs—doxorubicin (water soluble), silymarin (sparingly water soluble) and gliclazide (water insoluble)—and six polymers with varied functional groups—alginic acid, sodium alginate, chitosan, Gantrez AN119 (methyl-vinyl–ether-co-malic acid based), Eudragit L100 and Eudragit RSPO (both acrylic acid based)—were selected for the study. The structures were modeled and minimized using molecular mechanics force field (MM+). MD simulation (Gromacs-forcefield, 300 ps, 300 K) of the drug in the vicinity of the polymer molecule in the presence of water molecules was performed, and the interaction energy (IE) between them was calculated. This energy was evaluated with respect to electric-dipole, van der Waals and hydrogen bond forces. A good linear correlation was observed between IE and our own previous data on drug uptake^* [R ² = 0.65, R_adj² = 0.65,R_pre² = 0.56, {\hbox{R}}_{\rm{adj}}^2 = 0.65,{\hbox{R}}_{\rm{pre}}^2 = 0.56, and a F ratio of 30.25, P < 0.001; Devarajan et al. (2005) J Biomed Nanotechnol 1:1–9]. Maximum drug uptake by the polymeric nanoparticles (NP) was achieved in water as the solvent environment. Hydrophilic interaction between NP and water was inversely correlated with drug uptake. The MD simulation method provides a reasonable approximation of drug uptake that will be useful in developing polymer-based drug delivery systems.