Uddhavesh Sonavane

Study of Early Events in the Protein Folding of Villin Headpiece using Molecular Dynamics Simulation

Abstract 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.

Molecular dynamics simulations of cyclohexyl modified peptide nucleic acids (PNA).

Abstract Peptide Nucleic Acids (PNA) that bind sequence specifically to DNA/RNA are of major interest in the field of molecular biology and could form the basis for gene-targeted drugs. Molecular dynamics simulations are aimed to characterize the structural and dynamical features to understand the effect of backbone modification on the structure and dynamics along with the stability of the resulting 10mer complexes of PNA with DNA/RNA. Twelve Molecular Dynamics (MD) simulations of duplexes and triplexes with and without cyclohexyl modification were carried out for 10ns each. The simulations indicate that the cyclohexyl modification with different stereoisomers has influenced all the PNA-DNA/RNA complexes. Modification has added rigidity to backbone by restricting β to in case of (1R,2S) cyclohexyl PNA and to −60 in case of (1S,2R) cyclohexyl PNA. The results of MD simulations were able to show the backbone rigidification and preference for RNA complexes over DNA due to presence of cyclohexyl ring in the PNA backbone.

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

Multiscale modelling to understand the self-assembly mechanism of human β2-adrenergic receptor in lipid bilayer

The long perceived notion that G-Protein Coupled Receptors (GPCRs) function in monomeric form has recently been changed by the description of a number of GPCRs that are found in oligomeric states. The mechanism of GPCR oligomerization, and its effect on receptor function, is not well understood. In the present study, coarse grained molecular dynamics (CGMD) approach was adopted for studying the self-assembly process of the human GPCR, β2-adrenergic receptor (β2-AR), for which several experimental evidences of the dimerization process and its effect on cellular functions are available. Since the crystal structure of β2-AR lacks the third intracellular loop, initially it was modelled and simulated using restrained MD in order to get a stable starting conformation. This structure was then converted to CG representation and 16 copies of it, inserted into a hydrated lipid bilayer, were simulated for 10 μs using the MARTINI force field. At the end of 10μs, oligomers of β2-AR were found to be formed through the self-assembly mechanism which were further validated through various analyses of the receptors. The lipid bilayer analysis also helped to quantify this assembly mechanism. In order to identify the domains which are responsible for this oligomerization, a reverse transformation of the CG system back to all-atom structure and simulated annealing run were carried out at the end of 10 μs CGMD run. Analysis of the all-atom dimers thus obtained, revealed that TM1/TM1, H8/H8, TM1/TM5 and TM6/TM6 regions formed most of the dimerization surfaces, which is in accordance with some of the experimental observations and recent simulation results.

Conformational Preferences of Anticodon 3′-Adjacent Hypermodified Nucleic Acid Base cis-or trans-Zeatin and its 2-methylthio Derivative, cis-or trans-ms2Zeatin

Abstract Conformational preferences of the hypermodified nucleic acid bases N6-(Δ2 -cis-hydroxyisopentenyl)adenine, cis-io6Ade also known as cis-zeatin, and N6-(Δ2 -trans-hydroxyisopentenyl)adenine, trans-io6ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms2io6Ade or cis-ms2zeatin, and trans-ms2io6Ade or trans-ms2zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AMI and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io6Ade, trans-io6Ade, cis-ms2io6Ade and trans-ms2io6Ade 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-io6Ade as well as cis- ms2io6Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io6Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms2io6Ade, 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-ms2io6Ade as well as trans-ms2io6Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this nonobstrusive 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 i6Ade and ms2i6Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms2-zeatin (ms2io6Ade) modification.

Conformational flipping of the N(6) substituent in diprotonated N6‐(N‐glycylcarbonyl)adenines: The role of N(6)H in purine‐ring‐protonated ureido adenines

Conformational transitions of the N(6) substituent, in hypermodified nucleic acid base N6-(N-glycylcarbonyl)adenine, gc6Ade, on diprotonation of the adenine ring at any two of N(1), N(3), and N(7) sites, are studied using the quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. The N(6) substituent retains the usual “distal” orientation (α=0°) in (N(1),N(3)) diprotonated gc6Ade, but the “proximal” orientation (α=180°) is preferred instead, for (N(3),N(7)) and (N(7),N(1)) diprotonated gc6Ade. The proximal orientation may alter the reading frame during translation. Intramolecular N(6)HO(13b) hydrogen bonding is the key common feature, present in the preferred structure, for each of these variously diprotonated gc6Ade.

The remarkable efficiency of a Pin-II proteinase inhibitor sans two conserved disulfide bonds is due to enhanced flexibility and hydrogen bond density in the reactive site loop

Capsicum annuum (L.) expresses diverse potato type II family proteinase inhibitors comprising of inhibitory repeat domain (IRD) as basic functional unit. Most IRDs contain eight conserved cysteines forming four disulfide bonds, which are indispensible for their stability and activity. We investigated the functional significance of evolutionary variations in IRDs and their role in mediating interaction between the inhibitor and cognate proteinase. Among the 18 IRDs encoded by C. annuum, IRD-7, -9, and -12 were selected for further characterization on the basis of variation in their reactive site loop, number of conserved cysteine residues, and higher theoretical ΔGbind for interaction with Helicoverpa armigera trypsin. Moreover, inhibition kinetics showed that IRD-9, despite loss of some of the disulfide bonds, was a more potent proteinase inhibitor among the three selected IRDs. Molecular dynamic simulations revealed that serine residues in the place of cysteines at seventh and eighth positions of IRD-9 resulted in an increase in the density of intramolecular hydrogen bonds and reactive site loop flexibility. Results of the serine residues chemical modification also supported this observation and provided a possible explanation for the remarkable inhibitory potential of IRD-9. Furthermore, this natural variant among IRDs showed special attributes like stability to proteolysis and synergistic inhibitory effect on other IRDs. It is likely that IRDs have coevolved selective specialization of their structure and function as a response towards specific insect proteases they encountered. Understanding the molecular mechanism of pest protease–plant proteinaceous inhibitor interaction will help in developing effective pest control strategies. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:39

Conformational preferences of the base substituent in hypermodified nucleotide queuosine 5'-monophosphate 'pQ' and protonated variant 'pQH+'.

Abstract Conformational preferences of the base substituent in hypermodified nucleotide queuosine 5′-monophosphate ‘pQ’ and its protonated form ‘pQH+’ have been studied using quantum chemical Perturbative Configuration Interaction with Localized Orbitals PCILO method. The salient points have also been examined using molecular mechanics force field MMFF, parameterized modified neglect of differential overlap PM3 and Hartree Fock-Density Functional Theory HF DFT (pBP/DN*) approaches. Aqueous solvation of pQ and pQH+ has also been studied using molecular dynamics simulations. Consistent with the observed crystal structure, in isolated protonated form pQH+, the quaternary amine HN(13)+H, of the sidechain having 7-aminomethyl linkage, hydrogen bonds with the carbonyl oxygen O(10) of the base. However, N(13)H—0(10) hydrogen bonding is not preferred for unprotonated pQ, whether isolated or hydrated. Interaction between the 5′-phosphate and the 7-aminomethyl group is more likely for isolated pQ. The cyclopentenediol hydroxyl group 04″H may hydrogen bond with the 0(10) in isolated pQ as well as in pQH+. The 04″H may hydrogen bond with the 5′-phosphate as well. The presence of -CH2-NH- and 0″H groups in pQ and pQH+ allows interesting possibilities for intranucleotide hydrogen bonds and interactions across the anticodon loop. Simultaneous hydrogen bonds 02P—HN(13)+H—0(10) are indicated for hydrated pQH+. Unlike weak involvement of 04″H, these interactions also persist in hydrated pQH+ and may much reduce backbone flexibility. Resulting sub-optimal Q:C base pairing leads to unbiased reading of U or C as the third codon letter. Cyclopentenediol hydroxyl groups may interact with other biomolecules, allowing specific recognition. Prospective pQ34 and pQ34H+ sites for codon-anticodon base pairing remain unhindered, but non canonical Q:G base pairing (amber-suppression) is ruled out.

Insights into the folding pathway of the Engrailed Homeodomain protein using replica exchange molecular dynamics simulations

Protein folding studies were carried out by performing microsecond time scale simulations on the ultrafast/fast folding protein Engrailed Homeodomain (EnHD) from Drosophila melanogaster. It is a three-helix bundle protein consisting of 54 residues (PDB ID: 1ENH). The positions of the helices are 8-20 (Helix I), 26-36 (Helix II) and 40-53 (Helix III). The second and third helices together form a Helix-Turn-Helix (HTH) motif which belongs to the family of DNA binding proteins. The molecular dynamics (MD) simulations were performed using replica exchange molecular dynamics (REMD). REMD is a method that involves simulating a protein at different temperatures and performing exchanges at regular time intervals. These exchanges were accepted or rejected based on the Metropolis criterion. REMD was performed using the AMBER FF03 force field with the generalised Born solvation model for the temperature range 286-373 K involving 30 replicas. The extended conformation of the protein was used as the starting structure. A simulation of 600 ns per replica was performed resulting in an overall simulation time of 18 μs. The protein was seen to fold close to the native state with backbone root mean square deviation (RMSD) of 3.16 Å. In this low RMSD structure, the Helix I was partially formed with a backbone RMSD of 3.37 Å while HTH motif had an RMSD of 1.81 Å. Analysis suggests that EnHD folds to its native structure via an intermediate in which the HTH motif is formed. The secondary structure development occurs first followed by tertiary packing. The results were in good agreement with the experimental findings.

Way toward "dietary pesticides": molecular investigation of insecticidal action of caffeic acid against Helicoverpa armigera.

Bioprospecting of natural molecules is essential to overcome serious environmental issues and pesticide resistance in insects. Here we are reporting insights into insecticidal activity of a plant natural phenol. In silico and in vitro screening of multiple molecules supported by in vivo validations suggested that caffeic acid (CA) is a potent inhibitor of Helicoverpa armigera gut proteases. Protease activity and gene expression were altered in CA-fed larvae. The structure-activity relationship of CA highlighted that all the functional groups are crucial for inhibition of protease activity. Biophysical studies and molecular dynamic simulations revealed that sequential binding of multiple CA molecules induces conformational changes in the protease(s) and thus lead to a significant decline in their activity. CA treatment significantly inhibits the insects detoxification enzymes, thus intensifying the insecticidal effect. Our findings suggest that CA can be implicated as a potent insecticidal molecule and explored for the development of effective dietary pesticides.