Abstract

Improving thermostability of industrial enzymes is an important protein engineering challenge. Point mutations, induced to increase thermostability, affect the structure and dynamics of the target protein in several ways and thus can also affect its activity. There appears to be no general rules for improving thermostabilty of enzymes, without adversely affecting their enzymatic activity. We report MD simulations, of wild type Bacillus subtilis lipase (WT) and its six progressively thermostable mutants (2M, 3M, 4M, 6M, 9M and 12M), performed at different temperatures, to address this issue. Less thermostable mutants (LTMs), 2M - 6M, show WT-like dynamics at all simulation temperatures. However, the two more thermostable mutants (MTMs), show required flexibility at appropriate temperature ranges and maintain conformational stability at high temperature. They show deep and rugged free-energy landscape, confining them within a near-native conformational space by conserving non-covalent interactions, and thus protecting them from possible aggregation. In contrast, the LTMs having marginally higher thermostabilities than WT show greater probabilities of accessing non-native conformations, which, due to aggregation, have reduced possibilities of reverting to their respective native states under refolding conditions. Our analysis indicates the possibility of non-additive effects of point mutations on the conformational stability of LTMs.

Abstract

Hydrogen bonded non-canonical base pairs have been identified as the key player behind the structural complexity and functional diversity of the RNA molecule. Enormous research have been performed to understand the correlation between different base pairing geometries and their contributions in the overall structural stabilities and functionalities. Towards this goal, calculation of intrinsic interaction energy of different base pairing geometries using ab-initio and DFT based Quantum Mechanical methods provide a significant insight in the context of RNA. Analysis of deviation in geometry and intrinsic interaction energy between the crystal and ground state optimized structures reveal the chemical behaviours of a particular base pair and the contextual effects on its stability. Fueled by the advancement of post Hartree-Fock QM theories and more accurate basis sets, the present knowledge base is flooded with a large amount of QM analysis data which lacks the systematic organization and hence fails to serve the scientific community in the best possible way. We, therefore, have attempted to build a relational database, “RNA Base Pair Count Geometry and Stability (RNABP COGEST)”, which relates the occurrence frequencies of different base pairs in RNA with their crystal and ground state optimized geometries (calculated at different levels of QM theory) and corresponding intrinsic interaction energies. To the best of our knowledge, this is the first attempt to organize energetics information for different RNA base pairs along with their occurrences and geometries. Our database provides an interactive interface which enables the user to extract data by constructing diverse geometry, frequency and energy based search queries for the purpose of better understanding of the complex relation between the geometry and stability of RNA base pairs. RNABP COGEST is a relational database developed in MYSQL with PHP and HTML based web interface. Availability of consolidated QM analysis data regarding interaction energies, structural parameters, and occurrences of all putative base pairing geometries at a glance, in RNABP COGEST, along with multiple comparative searching options will definitely be useful to bridge the gap in the understanding of complex RNA structures and their functionalities

Abstract

Protonated nucleobases have significant roles in facilitating catalytic functions of RNA, and in stabilizing different structural motifs. Reported pKa values of nucleobase protonation suggest that the population of neutral nucleobases is 103 – 104 times higher than that of protonated nucleobases under physiological conditions (pH  7.4). Therefore, a molecular level understanding of various putative roles of protonated nucleobases cannot be achieved without addressing the question of how their occurrence propensities and stabilities are related to the free energy costs associated with the process of protonation under physiological conditions. With water as proton donor, we use advanced QM methods to evaluate the site specific protonation propensities of nucleobases in terms of their associated free energy changes (
Gprot). Quantitative follow up on the energetics of base pair formation and database search for evaluating their occurrence frequencies, reveal a lack of correlation between base pair stability and occurrence propensities on the one hand, and ease of protonation on the other. For example, although N7 protonated Adenine (
Gprot = 40.0 kcal/mol) is found to participate in stable base pairing, base pairs involving N7 protonated Guanine (
Gprot = 36.8 kcal/mol), on geometry optimization, converge to a minima where Guanine transfers its extra proton to its partner base. Such observations, along with examples of weak base pairs involving N3 protonation of Cytosine (
Gprot = 37.0 kcal/mol) are rationalized by analysing the protonation induced charge redistributions which are found to significantly influence, both positively and negatively, the hydrogen bonding potentials of different functional sites of individual nucloebases. Protonation induced charge redistribution is also found to strongly influence (i) the aromatic character of the rings of the participating bases and (ii) hydrogen bonding potential of the free edges of the protonated base pair. Comprehensive analysis of a non-redundant RNA crystal structure dataset further reveals that, while availability of stabilization possibilities determine the feasibility of occurrence of protonated bases, their occurrence context and specific functional roles are important factors determining their occurrence propensities.

Abstract

Efficiency of 6-ethynylpyridone (E), a potential Thymine (T) analogue, which forms high-fidelity base pairs with Adenine (A) and gives rise to stabler DNA duplexes, with stability comparable to those containing canonical Cytosine(C):Guanine(G) base pairs, has been reported recently. Estimates of the interaction energies, involving geometry optimization at the DFT level (including middle range dispersion interactions) followed by single point energy calculation at MP2 level, in excellent correlation with the experimentally observed trends, show that E binds more strongly and more discriminatively with A, than T does. Detailed analysis reveals that the increase in base-base interaction arises out of conjugation of acetylenic  electrons with the ring  system of E, which results in not only an extra stabilizing C–H· · ·  interaction in the EA pair, it also leads to a strengthening of the conventional hydrogen bonds. However, the computed base-base interaction energy for the EA pair was found to be much less than that of the canonical CG pair implying that the difference in the TA versus EA base pairing interaction alone cannot explain the large experimentally observed increase in the thermostability of DNA duplexes, where a TA pair is replaced with an EA pair. Our computations show that the conjugation of acetylenic  electrons with the ring  system also possibly plays a role in increasing the stacking potential of the EA pair, which in turn, can explain its marked influence in the enhancement of duplex stability.

Abstract

RNA structural bioinformatics has developed, into a vibrant interdisciplinary area, in response to the emergence of noncoding RNA molecules as key regulatory players in nearly all important biological processes. Here we introduce the different conceptual tools and available resources, related to this new area. We also review its application in studying the role of RNA-protein interactions.

Abstract

This paper proposes a method for automatic detection of breathy voiced vowels in continuous Gujarati speech. As breathy voice is a specific phonetic feature predominantly present in Gujarati among Indian languages, it can be used for identifying Gujarati language. The objective of this paper is to differentiate breathy voiced vowels from modal voiced vowels based on loudness measure. Excitation source characteristics represented by loudness measure are used for differentiating the voice quality. In the proposed method, initially vowel regions in continuous speech are determined by using the knowledge of vowel onset points and epochs. Later, hypothesized vowel segments are classified by using loudness measure. Performance of the proposed method is evaluated on Gujarati speech utterances containing around 47 breathy and 192 modal vowels spoken by 5 male and 5 female speakers. Classification of vowels into breathy or modal voice is achieved with an accuracy of around 94 %.

Abstract

In this paper, the non-uniform duration modification is exploited along with other prosody features for neutral speech to anger speech conversion. The non-uniform duration modification method modifies the durations of vowel and pause segments by different modification factors. Vowel segments are modified by factors based on their identities, and pause segments by uniform factors. Consonant and transition segments are not modified. These modification factors are derived from the analysis of neutral and anger speech. For this purpose, a well known Indian database named as the Indian Institute of Technology Kharagpur Simulated Emotion Speech Corpus (IITKGP-SESC) is chosen for analysis of emotions and synthesis of emotions from neutral speech. The prosodic features used in this study for emotion conversion are pitch contour, intensity contour, and duration contour. Subjective listening test results show that the effectiveness of perception of emotion is better in case of non-uniform duration modification than uniform duration modification

Abstract

DNA–RNA hybrids are heterogeneous nucleic acid duplexes consisting of a DNA strand and a RNA strand, and are formed as key intermediates in many important biological processes. They serve as substrates for the RNase H enzymatic activity, which has been exploited for several biomedical technologies such as antiviral and antisense therapies. To understand the relation of structural properties with the base composition in DNA–RNA hybrids, molecular dynamics (MD) simulations were performed on selected model systems by systematically varying the deoxypyrimidine (dPy) content from 0 to 100% in the DNA strand. The results suggest that the hybrid duplex properties are highly dependent on their deoxypyrimidine content of the DNA strand. However, such variations are not seen in their corresponding pure DNA and RNA duplex counterparts. It is also noticed that the systematic variation in deoxypyrimidine …

Abstract

Gold cluster–nucleobase complexes have potential applications in designing and fabrication of novel electronic nano-devices, and there has been a surge in research activities in this area recently. Binding of gold clusters (Au3 and Au4) with DNA bases and size-expanded DNA bases (x-bases) have been studied using density functional theory employing high quality basis set. A comprehensive attempt has been made to examine several gold–nucleobase complexes with respect to change in the orientation of Au clusters with respect to all the titratable sites of the bases. Geometric and electronic features of these complexes provided evidences for existence of non-conventional hydrogen bonds, which was further substantiated via vibrational frequency and natural bond orbital (NBO) analysis. The nucleobases, both canonical and size-expanded forms, form stable complexes with both the gold clusters considered …

Abstract

The viral protein U (Vpu) encoded by HIV-1 has been shown to assist in the detachment of virion particles from infected cells. Vpu forms cation-specific ion channels in host cells, and has been proposed as a potential drug target. An understanding of the mechanism of ion transport through Vpu is desirable, but remains limited because of the unavailability of an experimental structure of the channel. Using a structure of the pentameric form of Vpu – modeled and validated based on available experimental data – umbrella sampling molecular dynamics simulations (cumulative simulation time of more than 0.4 µs) were employed to elucidate the energetics and the molecular mechanism of ion transport in Vpu. Free energy profiles corresponding to the permeation of Na+ and K+ were found to be similar to each other indicating lack of ion selection, consistent with previous experimental studies. The Ser23 residue is shown to enhance ion transport via two mechanisms: creating a weak binding site, and increasing the effective hydrophilic length of the channel, both of which have previously been hypothesized in experiments. A two-dimensional free energy landscape has been computed to model multiple ion permeation, based on which a mechanism for ion conduction is proposed. It is shown that only one ion can pass through the channel at a time. This, along with a stretch of hydrophobic residues in the transmembrane domain of Vpu, explains the slow kinetics of ion conduction. The results are consistent with previous conductance studies that showed Vpu to be a weakly conducting ion channel.