6 research outputs found
Comparison of proteomic profiles of the venoms of two of the \u27Big Four\u27 snakes of India, the Indian Cobra (Naja naja) andthe common krait (Bungarus caeruleus), and analyses of their toxins
Snake venoms are mixtures of biologically-active proteins and peptides, and several studies have described the characteristics of some of these toxins. However, complete proteomic profiling of the venoms of many snake species has not yet been done. The Indian cobra (Naja naja) and common krait (Bungarus caeruleus) are elapid snake species that are among the ‘Big Four’ responsible for the majority of human snake envenomation cases in India. As understanding the composition and complexity of venoms is necessary for successful treatment of envenomation in humans, we utilized three different proteomic profiling approaches to characterize these venoms: i) one-dimensional SDS-PAGE coupled with in-gel tryptic digestion and electrospray tandem mass spectrometry (ESI-LC-MS/MS) of individual protein bands; ii) in-solution tryptic digestion of crude venoms coupled with ESI-LC-MS/MS; and iii) separation by gel-filtration chromatography coupled with tryptic digestion and ESI-LC-MS/MS of separated fractions. From the generated data, 81 and 46 different proteins were identified from N. naja and B. caeruleus venoms, respectively, belonging to fifteen different protein families. Venoms from both species were found to contain a variety of phospholipases A2 and three-finger toxins, whereas relatively higher numbers of snake venom metalloproteinases were found in N. naja compared to B. caeruleus venom. The analyses also identified less represented venom proteins including L-amino acid oxidases, cysteine-rich secretory proteins, 5’-nucleotidases and venom nerve growth factors. Further, Kunitz-type serine protease inhibitors, cobra venom factors, phosphodiesterases, vespryns and aminopeptidases were identified in the N. naja venom, while acetylcholinesterases and hyaluronidases were found in the B. caeruleus venom. We further analyzed protein coverage (Lys/Arg rich and poor regions as well as potential glycosylation sites) using in-house software. These studies expand our understanding of the proteomes of the venoms of these two medically-important species
Conformational Dynamics of thiM Riboswitch To Understand the Gene Regulation Mechanism Using Markov State Modeling and the Residual Fluctuation Network Approach
Thiamine pyrophosphate
(TPP) riboswitch is a cis-regulatory element
in the noncoding region of mRNA. The aptamer domain of TPP riboswitch
detects the high abundance of coenzyme thiamine pyrophosphate (TPP)
and modulates the gene expression for thiamine synthetic gene. The
mechanistic understanding in recognition of TPP in aptamer domain
and ligand-induced compactness for folding of expression platform
are most important to designing novel modulators. To understand the
dynamic behavior of TPP riboswitch upon TPP binding, molecular dynamics
simulations were performed for 400 ns in both apo and TPP bound forms
of thiM riboswitch from <i>E. coli</i> and analyzed in terms
of eRMSD-based Markov state modeling and residual fluctuation network.
Markov state models show good correlations in transition probability
among metastable states from simulated trajectory and generated models.
Structural compactness in TPP bound form is observed which is correlated
with SAXS experiment. The importance of junction of P4 and P5 is evident
during dynamics, which correlates with FRET analysis. The dynamic
nature of two sensor forearms is due to the flexible P1 helix, which
is its intrinsic property. The transient state in TPP-bound form was
observed in the Markov state model, along with stable states. We believe
that this transient state is responsible to assist the influx and
outflux of ligand molecule by creating a solvent channel around the
junction region of P4 and P5 and such a structure was anticipated
in FRET analysis. The dynamic nature of riboswitch is dependent on
the interaction between residues on distal loops L3 and L5/P3 and
junction P4 and P5, J3/2 which stabilize the J2/4. It helps in the
transfer of allosteric information between J2/4 and P3/L5 tertiary
docking region through the active site residues. Understanding such
information flow will benefit in highlighting crucial residues in
highly dynamic and kinetic systems. Here, we report the residues and
segments in riboswitch that play vital roles in providing stability
and this can be exploited in designing inhibitors to regulate the
functioning of riboswitches
Identification of novel natural inhibitor for NorM – a multidrug and toxic compound extrusion transporter – an <i>insilico</i> molecular modeling and simulation studies
<p>The emergence of bacterial multidrug resistance is an increasing problem in treatment of infectious diseases. An important cause for the multidrug resistance of bacteria is the expression of multidrug efflux transporters. The multidrug and toxic compound extrusion (MATE) transporters are most recently recognized as unique efflux system for extrusion of antimicrobials and therapeutic drugs due to energy stored in either Na<sup>+</sup> or H<sup>+</sup> electrochemical gradient. In the present study, high throughput virtual screening of natural compound collections against NorM – a MATE transporter from <i>Neisseria gonorrhea</i> (NorM-NG) has been carried out followed by flexible docking. The molecular simulation in membrane environment has been performed for understanding the stability and binding energetic of top lead compounds. Results identified a compound from the Indian medicinal plant “Terminalia chebula” which has good binding free energy compared to substrates (rhodamine 6 g, ethidium) and more favorable interactions with the central cavity forming active site residues. The compound has restricted movement in TM7, TM8, and TM1, thus blocking the disruption of Na+ – coordination along with equilibrium state bias towards occlude state of NorM transporter. Thus, this compound blocks the effluxing pathway of antimicrobial drugs and provides as a natural bioactive lead inhibitor against NorM transporter in drug-resistant gonorrhea.</p