Abstract

Protonation and deprotonation of RNA bases, although energetically unfavorable, are known to play a key role in stabilization of the global structure of the RNA molecules and in regulating their functionalities.1 It is expected that protonation/deprotonation induced geometric changes and charge redistribution, in the concerned bases, govern their consequent stabilization through base pairing and other noncovalent interactions. We have used QM methods to look at these aspects, along with their gas phase and solvent phase energies, for individual, protonated and deprotonated bases. Geometry optimization and energy calculations were carried out at different levels of theory (B3LYP/6-31G+ +(2d,2p) and MP2/aug-ccpvdz) and solvent phase calculations were performed using CPCM polarizable conductor calculation model. Protonation/deprotonation processes have been modeled as isodesmic reactions considering water as proton acceptor/donor. It has been observed that protonation and deprotonation at all possible sites are disfavored processes characterized by a positive free energy change. It is however important to note that protonation at any exocyclic site or at ring imino nitrogen sites, and deprotonation from any site lead to planar and stable geometry with a decrease in entropy of the corresponding conjugate acids and bases respectively. Comparison of the geometrical parameters and Mulliken charge distribution in the neutral bases, with those of the corresponding charged species, showed interesting variations depending on the identity of the bases as well as on their respective protonation/deprotonation sites. The HOMOLUMO band gap has been found to increase on protonation and decrease on deprotonation. In solvent phase, a decrease in the pka values has been observed indicating the increased stabilization of the charged species in polar environment. This study can be useful in understanding the structure and dynamics of RNA under pH strain.

Abstract

Molecular level understanding of physiochemical interactions responsible for the structure and dynamics of functional RNA is a major challenge to the scientific community. RNA structural diversity is mainly due to the presence of non-canonical base-pairs, phosphate mediated interactions and interactions between two or more nucleotides involving water molecules and ions that stabilize folded RNA structures. BPFIND and INCAR are two complementary tools developed to analyse RNA 3D structure in terms of hydrogen-bonded secondary structural elements interacting through primarily non-canonical tertiary base pairing interactions. Identification of recurring combinations of such elements or motifs, important in terms of architecture enhances our understanding of the RNA tertiary structures. First tool, Base Pair Finder (BPFIND), detects and analyzes all canonical and non-canonical basepairs and base triples involving at least two direct hydrogen bonds formed between polar atoms of the bases or sugar O2’ only. Apart from detection, the program also gives a quantitative estimate of the conformational deformation of detected base pairs in comparison to an ideal planar base pair, which helps to understand their specific role towards structural and functional diversity. This approach is constrained by its dependence on identification of strongly hydrogen bonded base pairs only. The second tool, namely INCAR (Inter Nucleotide Contact Annotator in RNA), compensates this limitation by following a bottom-up approach towards identifying and characterizing structural features. At the lower end it detects, all inter-nucleotide hydrogen bonding interactions (both strong and weak), water and ion mediated interactions. At the other end it can capture the recurrent structural motifs made of several base-pairs and higher order interactions. This python based automated tool efficiently manages space and time complexity to capture, categorize and suitably annotate all significant non-covalent interactions, within a non-redundant dataset of RNA crystal structures. Along with this we have developed a visualization tool, VASTERN, which helps users to view the backbone structure as well as base pairs to analyze the structure interactively.

Abstract

Lipases are used in several industrial processes and thermostable lipases are very important in the detergent industry and in organic synthesis. B. subtilis lipase (Lip A) is a monomeric protein without any bound ligand. Lip A preferentially hydrolyses C8 fatty acid esters and it is a "lidless" lipase that does not show interfacial activation [1]. The mechanisms of thermostability of lipases are varied and depend on type of lipase; nevertheless, some common features that contribute to thermostability include additional non-covalent interactions, increase in packing density of the hydrophobic core, stabilization of alpha-helix and shortening of loops between secondary structural elements [3]. We have studied wild type Lip A along with its five thermostable mutants derived from wild type by introducing 2, 3, 4, 6 and 9 point mutations respectively through directed evolution. Experimentally, it was observed that the thermostabililty of the mutant lipases increased with the number of mutations introduced, without affecting their catalytic activity at mesophilic temperatures and the most thermostable mutant with nine mutations shows remarkable enhancement in melting temperature and temperature optima of activity compared to wild type [2]. Long time molecular dynamics (MD) simulations performed at both low and high temperatures, have provided us molecular-level insights into how point mutations can affect protein structure and dynamics. From our simulations we observed a reduced rmsd in several key regions, an increased overall flexibility with decrease in flexibility in stabilizing regions, an increase in the number of hydrogen bonds, decrease in SASA of hydrophobic regions for the most thermostable mutant lipase. Through PCA analysis we also show that the most thermostable lipase show a C-terminal stabilization through reduced fluctuation, which was not observed for the wild type and other less thermostable mutant lipases. However, thermostable lipases must retain their threedimensional structures and required flexibility at high temperatures in order to retain activity. The encouraging outcome suggests that the high-temperature MD simulations and the structure–thermostability relationships may be considered as a valuable tool for understanding the thermostability of lipases.

Abstract

Independent folding units which have the capability of carrying out biological functions have been classified as “protein domains”. These minimal structural units lead not only to considerable sequence changes of protein domains of similar folds and functions, but also gives rise to remarkable length variations under evolutionary pressure. Rapid and heuristic sequence search algorithms are generally sensitive and effective in recognizing protein domains that are distantly related within large sequence databases, but are not well-suited to identify remote homologues of varying lengths. An even more challenging aspect is introduced to distinguish reliable hits from a vast number of putative false positives that could have suboptimal sequence similarities. Here, the authors present a data-mining approach that provides stage-specific filters in sequence searches to reliably accumulate remote homologues, which encourages sampling of length variations albeit with a low false positive rate. Realization of such remote homologues with vivid length variations could contribute to better understanding of functional variety within protein domain superfamilies.

Abstract

In some bacteria, the intracellular concentration of several amino acids is controlled by riboswitches. One class of such riboswitches is Thermotoga maritima lysine-responsive riboswitch. It regulates the downstream expression of aspartate-semialdehyde dehydrogenase, which is involved in synthesis of precursor for methionine, threonine, lysine and diaminopimelate, depending on the presence or absence of small metabolite lysine. The GC-rich lysine riboswitch is among the longest and most complexly folded metabolite-sensing aptamers. Crystal structures of lysine sensing domain of riboswitch in presence and absence of lysine, feature a complex architecture, involving a modified four-way junction (J1-2, J2-3, J4-5 and J5-1) organized between coaxial bundles of three helices (P2, P3, P4) above and two helices (P1, P5) below. An intriguing structure to visualize is that adopted by P2, a long helix, which experiences two turns to have kissing-loop interactions between L2 and L3. The binding pocket of metabolite lysine is compact, bounded by the modified four-way junction, where lysine is specifically recognized through hydrogen bonds to its charged ends. In order to investigate the dynamics and conformational changes associated with the ligand binding, at the molecular level, we have carried out all-atom explicit solvent molecular dynamics simulations of aptamer domain of Thermotoga maritima asd lysine riboswitch in lysine-bound (HOLO) and lysine-free (APO) states using two widely accepted nucleic acids forcefields: CHARMM 27 and AMBER ff99parmbsc0. Detailed analysis, including principal component analysis and conformational clustering of the trajectories, reveal interesting insights into changes in conformational dynamics accompanying ligand binding. While confirming the existence of a preformed and ‘binding ready’ APO form and ligand binding induced stabilization of the expression platform linking P1 helix, our studies indicate importance of two tertiary interactions: loop-loop L2-L3 and helix-loop P2-L4 interactions in the functioning of lysine riboswitch, relating their role in stabilizing the aptamer domain. Our simulations also provide molecular insights into ligand binding mechanism by which lysine interacts with binding pocket to form a stable HOLO lysine riboswitch aptamer.

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

Vpu is an 81-residue protein encoded by human immunodeficiency virus type I (HIV-1) that facilitates viral release from host cells. The protein has a cytoplasmic domain and a helical transmembrane (TM) domain, of which the latter oligomerizes to form cation-specific ion channels. The number of TM domains that constitute the channel is still unclear, with experimental studies indicating the existence of a variety of oligomeric states. In this study, we have examined the possibility of Vpu to exist in tetra-, penta-, and hexameric states using comprehensive molecular dynamics (MD) simulations. By modeling the TM domain as an ideal α-helix, we carried out replica-exchange MD simulations in an implicit membrane environment for obtaining suitable starting structures, which were then subjected to extensive MD simulations in a fully hydrated lipid bilayer environment. The results show that the pentameric form is the most stable oligomeric state (the tetramer and hexamer models lose their initial channel-like structure), with helices in the pentamer being held together by strong van der Waals interactions. Hydrogen bonds between lipid headgroups and basic/hydrophilic residues on the protein are stronger in the pentamer than in the tetramer or the hexamer, indicating that these interactions might play a role in adhering the pentamer to the membrane. Free energy calculations using umbrella sampling technique have been performed to examine the potassium ion permeation through the pentameric pore. The results show a high free energy barrier corresponding to ion transport indicating weak ion channel activity in agreement with previous experimental biophysical studies.

Abstract

Pipeline generally extends over long distances traversing through wide variety of different soils, geological conditions and regions with different seismicity. Majority of the past works in the area of pipeline subjected to fault motion is restricted in several ways. There were many analytical models developed in the past for pipeline fault crossing, however, they are of limited usage, for example analytical model developed for pipeline fault crossing can be useful for strike slip fault crossing only. Likewise incorporating the large geometric changes in analytical study is a tricky task; however pipeline subjected to the fault motion itself is a phenomenon of large geometric changes. Especially when pipeline subjected to compression, where in addition to material deformation it also undergoes general as well as local buckling with bending, contradictorily past work mostly assumed that pipeline is under tension. With day by day increasing capacity of computation and advancement in numerical modeling, one can find more facts for pipeline subjected fault motions including cases of pipe under compression as well. In this paper, past work is reviewed for pipeline subjected to large fault motion. A three dimensional FE based numerical model is suggested to carry out pipeline performance of buried pipeline subjected to fault motion. A proposed model includes material nonlinearity, as well as effect of the large geometric changes. For this purpose, three dimensional FE program is developed in MATLAB. Displacement controlled Arc-length technique is implemented to solve the nonlinear behavior. To reduce the computation time of analysis here parallelization tool kit of MATLAB is utilized.

Abstract

Reinforced concrete buildings constitute the dominant type of construction in the earthquake prone countries. Many researchers have studied the performance of the RC structure in seismic environment. In this study a RC building of four storeys is considered and its performance is evaluated when subjected to five ground motions. The study is roughly divided into three parts, first part includes investigation of the dynamic characteristics of ground excitations, second part includes evaluation of the nonlinear seismic behaviour of building subjected to the given ground excitations and third part includes evaluation of damage. The present study is based on analytical investigation of seismic performance and potential seismic damage of a reinforced concrete framed building due to earthquakes in India, using nonlinear modelling and displacement-based analysis techniques. Since the seismic damage is directly correlated to the displacement (deformation) of the structure, the yield displacement of the structure is evaluated from the pushover analysis. From the displacement time history of the structure response, the peak values which exceeding yield displacement of the structure have been identified and the hysteresis energy is calculated for each cycle to calculate the damage of the structure. The estimation of structural damage is calculated as per the model of Park and Ang. At the end, authors attempted to propose a new damage scale.

Abstract

Pipeline generally extends over long distances traversing through wide variety of soil conditions with different seismicity. Majority of past work mainly restricted to pipelines exposed to strike slip fault motion or transverse permanent ground deformation that too cases of pipelines under tension. The study of buried pipeline under compression is of extreme importance, especially pipelines passing through thrusting sub-continent like India, where majority of pipelines are exposed to compression. In compression pipelines fail in material as well as in geometry. In this paper past work is reviewed and 3D finite element based model is proposed to compare pipeline performance subjected to different types of ground motion. The proposed model includes material non-linearity as well as effect of large geometric changes. In this proposed model displacement control Arc-length technique is implemented to solve the nonlinear behavior. To reduce the computational time of analysis a parallelization tool kit of MATLAB is utilized.