[show abstract][hide abstract] ABSTRACT: We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60-E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60-62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.
Biochimica et Biophysica Acta 12/2013; 1834(12):2736–2749. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: With the potential to engineer biological systems, synthetic biology is an emerging field that combines various disciplines of sciences. It encompasses combinations of DNA, RNA and protein modules for constructing desired systems and the "rewiring" of existing signalling networks. Despite recent advances, this field still lags behind in the artificial reconstruction of cellular processes, and thus demands new modules and switches to create "genetic circuits". The widely characterised noncanonical nucleic acid secondary structures, G-quadruplexes are promising candidates to be used as biological modules in synthetic biology. Structural plasticity and functional versatility are significant G-quadruplex traits for its integration into a biological system and for diverse applications in synthetic circuits.
[show abstract][hide abstract] ABSTRACT: Bacillus anthracis Ser/Thr protein kinase PrkC (BasPrkC) is important for virulence of the bacterium within the host. Homologs of PrkC and its cognate phosphatase PrpC (BasPrpC) are the most conserved mediators of signaling events in diverse bacteria. BasPrkC homolog in Bacillus subtilis regulates critical processes like spore germination and BasPrpC modulates the activity of BasPrkC by dephosphorylation. So far, biochemical and genetic studies have provided important insights into the roles of BasPrkC and BasPrpC; however, regulation of their activities is not known. We studied the regulation of BasPrkC/BasPrpC pair and observed that Zn(2+) metal ions can alter their activities. Zn(2+) promotes BasPrkC kinase activity while inhibits the BasPrpC phosphatase activity. Concentration of Zn(2+) in growing B. anthracis cells was found to vary with growth phase. Zn(2+) was found to be lowest in log phase cells while it was highest in spores. This variation in Zn(2+) concentration is significant for understanding the antagonistic activities of BasPrkC/BasPrpC pair. Our results also show that BasPrkC activity is modulated by temperature changes and kinase inhibitors. Additionally, we identified Elongation Factor Tu (BasEf-Tu) as a substrate of BasPrkC/BasPrpC pair and assessed the impact of their regulation on BasEf-Tu phosphorylation. Based on these results, we propose Zn(2+) as an important regulator of BasPrkC/BasPrpC mediated phosphorylation cascades. Thus, this study reveals additional means by which BasPrkC can be activated leading to autophosphorylation and substrate phosphorylation.
[show abstract][hide abstract] ABSTRACT: Loop length, loop composition, salt concentration and number of G-quartets are major determinants of G-quadruplex stability. We examined the effect of each of these factors on the thermal stability and folding topology of a library of RNA quadruplexes. Thermal stability of G2 and G3 RNA quadruplexes were investigated upon varying the loop length (from 1-1-1 to 15-15-15) and salt concentration (1 mM to 100 mM KCl) while the effect of loop composition was explored using eighteen naturally occurring potential RNA quadruplexes predicted in untranslated regions (UTRs). We found loop length and quadruplex stability to be inversely related for G2 RNA quadruplexes and G3 RNA quadruplexes with shorter loops. However, melting temperature saturates for G3 RNA quadruplexes with longer loops. RNA G-quadruplexes with longer loops (G3 15-15-15) displayed Tm value significantly higher than the physiological temperature. This study thus highlights the need to modify the consensus motif presently used by quadruplex prediction tools. An increase in the loop size from 7 bases to 15 bases in the consensus motif will add to its predictive value for the discovery of potential RNA quadruplexes across transcriptomes.
The Journal of Physical Chemistry B 05/2013; · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: RNA-binding proteins are an important class of mediators that regulate cell function and differentiation. Methylation of arginine, a post-translational modification (PTM) found in these proteins, can modulate their function. Arginine can be monomethylated or dimethylated, depending on the type of methyltransferases involved. This paper describes a comparative study of the thermodynamics of unmodified and modified Tat peptide interaction with TAR RNA, where the peptide is methylated at epsilon (ε) and eta (η) nitrogen atoms of guanidinium group of arginine side chain at position 52 or 53. The results indicate that monomethylation of arginine at epsilon (ε) nitrogen atom enhances binding affinity, owing to a more favourable enthalpy component which overrides the less favourable entropy change. In contrast, monomethylation of arginine residue at η nitrogen results in reduced binding affinity originating exclusively from a less favourable enthalpy change leaving entropic component unaffected. However, in case of simultaneous methylation at ε and η positions, the binding parameters remain almost unaffected, when compared to the unmodified peptide. In case of symmetric dimethylation at η position the observed enthalpy change of the binding was found to be smaller than the values obtained for the unmodified peptide. Asymmetric dimethylation at η position showed the most reduced binding affinities owing to less favourable enthalpy changes. These results provide insights that enable elucidation of the biological outcome of arginine methylation as PTMs that regulate protein function, and will contribute to our understanding of how these PTMs are established in vitro and in vivo.
[show abstract][hide abstract] ABSTRACT: miRNAs are small non-coding RNAs that regulate about 60% of mammalian genes by modulating their transcript levels. Network scale studies of miRNA-mediated regulatory circuits demonstrate the central importance of this class of small RNA in the maintenance of biological robustness. More recently, several reports have described the deregulation of numerous miRNA to be causally associated with many diseases, including cancer. These studies have highlighted the potential for development of therapeutic modalities against miRNA. Previous screening protocols, for small molecules targeting miRNA function, are either costly or technically too complex to be applied in a high-throughput manner in standard chemical laboratories. We describe a simple in vitro screening method using a DNA-based molecular beacon that overcomes the limitations associated with earlier screens. We used this method to identify inhibitors of miR-27a function from a library of 14 aminoglycosides as a pilot study. Inhibitory molecules identified were further scrutinized to identify the validity of screen. With this proof of concept we illustrate the utility of a scalable molecular-beacon-based screening strategy for miRNA inhibitors.
ACS Chemical Biology 03/2013; · 5.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transforming Growth Factor β2 (TGFβ2) is a versatile cytokine with prominent role in cell migration, invasion, cellular development and immunomodulation. TGFβ2 promotes the malignancy of tumors by inducing epithelial-mesenchymal transition (EMT), angiogenesis and immunosuppression. As it is well documented that nucleic acid secondary structure can regulate gene expression, we assessed whether any secondary motif regulates its expression at post transcriptional level. Bioinformatics analysis predicts an occurrence of 23 nucleotides putative G-quadruplex sequence (PG4) in the 5' UTR of TGFβ2 mRNA. The ability of this stretch of sequence to form a highly stable, intramolecular parallel quadruplex was demonstrated using UV and CD spectroscopy. Footprinting studies further validated its existence in presence of neighboring nucleotide sequence. Following structural characterization, we evaluated the biological relevance of this secondary motif using dual luciferase assay. Although PG4 inhibits the expression of the reporter gene, its presence in context to entire 5' UTR sequence interestingly enhances the gene expression. Mutation or abolition of G-quadruplex sequence from the 5' UTR of the gene diminished the expression of this gene at the translational level. Thus, here we highlight an activating role of G-quadruplex in modulating gene expression of TGFβ2 at the translational level and its potential to be used as a target for development of therapeutics against cancer.
[show abstract][hide abstract] ABSTRACT: The role of bacterial DnaJ protein as a co-chaperone of DnaK is well appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In the current study, we demonstrate that DnaJ binds a model non-native substrate with low nanomolar dissociation constant and more importantly, modulate the structure of the non-native state differently from DnaK/J complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we deciphered that the zinc-binding motif together with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that, this unforeseen structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.
[show abstract][hide abstract] ABSTRACT: We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60–E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60–62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.
[show abstract][hide abstract] ABSTRACT: Post-translational modification (PTM) of RNA binding proteins (RBPs) play a very important role in determining their binding to cognate RNAs and therefore regulate the downstream effects. Lysine can undergo various PTMs and thereby contribute to the regulation of different cellular processes. It can be reversibly acetylated and methylated using a pool of respective enzymes, to act as a switch for controlling the binding efficiency of RBPs. Here we have delineated the thermodynamic and kinetic effects of N-acetylation and N-monomethylation of lysine on interaction between HIV-1 TAR RNA and its cognate binder Tat peptide ( a model system). Our results indicate that acetylation of lysine 50 (K50), leads to eight- fold reduction in binding affinity, originating exclusively from entropy changes whereas, lysine 51 (K51) acetylation resulted only in three fold decrease with large enthalpy-entropy compensation. The measurement of kinetic parameters indicated major change (4.5 fold) in dissociation rate in case of K50 acetylation however, K51 acetylation showed similar effect on both association and dissociation rates. In contrast, lysine methylation did not affect the binding affinity of Tat peptide to TAR RNA at K50, nonetheless three fold enhancement in binding affinity was observed at K51 position. In spite of large enthalpy-entropy compensation, lysine methylation seems to have more pronounced position specific effect on the kinetic parameters. In case of K50 methylation, simultaneous increase was observed in the rate of association and dissociation leaving binding affinity unaffected. The increased binding affinity for methylated Tat at K51 stems from faster association rate with slightly slower dissociation rate.
PLoS ONE 01/2013; 8(10):e77595. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The interaction of the trans-activation responsive (TAR) region of bovine immunodeficiency virus (BIV) RNA with the Tat peptide is known to play important role in viral replication. Despite being thoroughly studied through a structural point of view, the nature of binding between BIV TAR RNA and the BIV Tat peptide requires information related to its thermodynamics and the nature of hydration around the TAR-Tat complex. In this context, we carried out the thermodynamic study of binding of the Tat peptide to the BIV TAR RNA hairpin through different calorimetric and spectroscopic measurements. Fluorescence titration of 2-aminopurine tagged BIV TAR RNA with the Tat peptide gives their binding affinity. The isothermal titration calorimetric experiment reveals the enthalpy of binding between BIV TAR RNA and the Tat peptide to be largely exothermic with the value of -11.7 (SEM 0.2) kcal mol(-1). Solvation dynamics measurements of BIV TAR RNA having 2-AP located at the bulge region have been carried out in the absence and presence of the BIV Tat peptide using the time correlated single photon counting technique. The solvent cage around the Tat binding site of RNA appears to be more rigid in the presence of the Tat peptide as compared to the free RNA. The displacement of solvent and ions on RNA due to peptide binding influences the entropic contributions to the total binding energy.
[show abstract][hide abstract] ABSTRACT: Human telomeric DNA has the ability to fold into a 4-stranded G-quadruplex structure. Several G-quadruplex ligands are known to stabilize the structure and thereby inhibit telomerase activity. Such ligands have demonstrated efficient telomerase inhibition in dilute conditions, but under molecular crowding conditions mimicking physiological milieu, stabilization of the telomeric G-quadruplex is often lost. We attempted to demonstrate the enhanced G-quadruplex stabilizing ability under molecular conditions by using twisted intercalating nucleic acids (TINA)-modified oligonucleotides. We have shown using circular dichroism and ultraviolet spectroscopic methods that these TINA-modified short oligonucleotides function as G-quadruplex, inducing agents and participate in the formation of stabilized 3:1 G-quadruplex with the human telomeric oligonucleotide. Using enzyme-linked immunosorbent assay-based telomerase repeat amplification assay (TRAP) assay as well as nondenaturing polyacrylamide gel electrophoresis-based TRAP, we demonstrate remarkable enhancement in their anti-telomerase activity even under molecular crowding conditions. This is the first time in which a G-quadruplex stabilizing agent has demonstrated enhanced activity even under molecular crowding conditions.
[show abstract][hide abstract] ABSTRACT: Cell selective, naturally occurring, host defence cationic peptides present a good template for the design of novel peptides with the aim of achieving a short length with improved antimicrobial potency and selectivity. A novel, short peptide CS-1a (14 residues) was derived using a sequence hybridization approach on sarcotoxin I (39 residues) and cecropin B (35 residues). The sequence of CS-1a was rearranged to enhance amphipathicity with the help of a Schiffer-Edmundson diagram to obtain CS-2a. Both peptides showed good antibacterial activity in the concentration range 4-16 μg·mL(-1) against susceptible as well as drug-resistant bacterial strains, including the clinically relevant pathogens Acenatobacter sp. and methicillin-resistant Staphylococcus aureus. The major thrust of these peptides is their nonhaemolytic activity against human red blood cells up to a high concentration of 512 μg·mL(-1) . Compared to CS-1a, amphipathic peptide CS-2a showed a more pronounced α-helical conformation, along with a better membrane insertion depth in bacterial mimic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) small unilamellar vesicles. With equivalent lipid-binding affinity, the two peptides assumed different pathways of membrane disruption, as demonstrated by calcein leakage and the results of transmission electron microscopy on model bacterial mimic large unilamellar vesicles. Extending the work from model membranes to intact Escherichia coli cells, differences in membrane perturbation were visible in microscopic images of peptide-treated E. coli. The present study describes two novel short peptides with potent activity, cell selectivity and divergent modes of action that will aid in the future design of peptides with better therapeutic potential.
[show abstract][hide abstract] ABSTRACT: Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones.
[show abstract][hide abstract] ABSTRACT: Dual specificity protein kinases (DSPKs) are unique enzymes that can execute multiple functions in the cell, which are otherwise performed exclusively by serine/threonine and tyrosine protein kinases. In this study, we have characterized the protein kinases Bas2152 (PrkD) and Bas2037 (PrkG) from Bacillus anthracis. Transcriptional analyses of these kinases showed that they are expressed in all phases of growth. In a serendipitous discovery, both kinases were found to be DSPKs. PrkD was found to be similar to the eukaryotic dual specificity Tyr phosphorylation-regulated kinase class of dual specificity kinases, which autophosphorylates on Ser, Thr, and Tyr residues and phosphorylates Ser and Thr residues on substrates. PrkG was found to be a bona fide dual specificity protein kinase that mediates autophosphorylation and substrate phosphorylation on Ser, Thr, and Tyr residues. The sites of phosphorylation in both of the kinases were identified through mass spectrometry. Phosphorylation on Tyr residues regulates the kinase activity of PrkD and PrkG. PrpC, the only known Ser/Thr protein phosphatase, was also found to possess dual specificity. Genistein, a known Tyr kinase inhibitor, was found to inhibit the activities of PrkD and PrkG and affect the growth of B. anthracis cells, indicating a possible role of these kinases in cell growth and development. In addition, the glycolytic enzyme pyruvate kinase was found to be phosphorylated by PrkD on Ser and Thr residues but not by PrkG. Thus, this study provides the first evidence of DSPKs in B. anthracis that belong to different classes and have different modes of regulation.
Journal of Biological Chemistry 06/2012; 287(32):26749-63. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this work, the binding kinetics of amino acid-based surfactants, presenting different linkers and head groups, with calf thymus (CT)-DNA was studied using stopped-flow fluorescence spectroscopy. The kinetic studies were carried out as a function of Na(+) concentration and surfactant-to-DNA charge ratio. The surfactant binding on DNA took place in two consecutive steps, for which the corresponding first and second relative rate constants (k(1) and k(2)) were determined. The fast step was attributed to the surfactant binding to DNA and micelle formation in its vicinity, the slower step to DNA condensation and possible rearrangement of the surfactant aggregates. In general, both relative rate constants increase with surfactant concentration and decrease with the ionic strength of the medium. The architecture of the surfactant was found to have a significant impact on the kinetics of the DNA-surfactant complexation. Surfactants with amide linkers showed larger relative rate constants than those with ester linkers. The variation of the relative rate constants with the head groups of the surfactants, alanine and proline, was found to be less obvious, being partially dependent on the surfactant concentration.
The Journal of Physical Chemistry B 05/2012; 116(20):5831-7. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: MicroRNAs (miRNAs) have crucial functions in many cellular processes, such as differentiation, proliferation and apoptosis; aberrant expression of miRNAs has been linked to human diseases, including cancer. Tools that allow specific and efficient knockdown of miRNAs would be of immense importance for exploring miRNA function. Zebrafish serves as an excellent vertebrate model system to understand the functions of miRNAs involved in a variety of biological processes. We designed and employed a strategy based on locked nucleic acid enzymes (LNAzymes) for in vivo knockdown of miRNA in zebrafish embryos. We demonstrate that LNAzyme can efficiently knockdown miRNAs with minimal toxicity to the zebrafish embryos.
[show abstract][hide abstract] ABSTRACT: G-quadruplexes are non canonical secondary structures held together by Hoogsteen bonded planar guanine quartets formed in G-rich sequences in DNA and RNA. Considerable research over the past three decades has contributed to a great deal of understanding of these unusual structures in DNA. Various factors governing the stability of DNA quadruplexes coupled with their in vivo existence have been well documented. RNA has emerged as a key regulatory player in the functioning of the cell shifting the focus to RNA G-quadruplexes which were discovered recently. RNA G-quadruplexes demonstrate immense potential for in vivo existence and function due to their inherent chemistry. We have highlighted the major findings of the field and compared them to structural aspects of DNA quadruplexes. Further, the plausible functions of RNA G-quadruplexes such as translational suppression, splicing etc. are discussed in brief, suggesting scope for an extensive role of these structures in biological systems. As the field is growing, we endeavor to review the current knowledge and evaluate the various attributes of RNA G- quadruplex structure, stability, function and applications. We have also attempted to evaluate the physical and physiological role and relevance of these motifs.
Current pharmaceutical design 02/2012; 18(14):2102-11. · 4.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: DNA G-quadruplexes are known as modulators of transcription. More recently G-quadruplexes, located in the untranslated regions of the mRNA of protein coding genes, have been described to negatively regulate gene expression at the post transcriptional/ translational levels. Here we describe the possibility of the existence of G-quadruplexes in non-coding RNA (ncRNA) and discuss their potential biological roles. Using an in house prediction tool (Quadfinder) we observe a significant occurrence and distribution of G-quadruplexes in ncRNA of various sizes. We also observe that most of non-coding RNAs harboring these potential quadruplex motifs peak at the sizes ranging from 200-300 bases. More importantly we report enrichment for single and dinucleotide loops indicating a degree of high stability of these G-quadruplexes and their potential functions in vivo. Subsequent in vitro analyses of a subset of these sequences were performed which support our predictions.
[show abstract][hide abstract] ABSTRACT: No dice: MicroRNAs (miRNAs) fine-tune gene expression, deregulation of which has been causally associated with a number of debilitating conditions. Streptomycin, a well-known aminoglycoside drug, binds to RNA secondary structures and is shown to inhibit miR-21 function by direct binding to its precursor, thus presumably interfering with the processing by the Dicer enzyme.
Angewandte Chemie International Edition 12/2011; 51(4):1019-23. · 13.73 Impact Factor