April 11, 2014 at 2.30 pm in AG-80
Molecular Machinery Involved in the Process of Small RNA Mediated Gene Regulation
Micro-RNA (miRNA) and small interfering-RNA (siRNA) are short (~22-nucleotide), single-stranded RNA molecules that regulate gene expression by promoting degradation or translational inhibition of target mRNAs. They influence diverse biological functions through the repression of target genes during normal development and pathological responses. A hallmark of small-RNA mediated gene silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. The current model suggests that Dicer selects cleavage sites by measuring a set distance from the 3′ overhang of the double-stranded RNA terminus. Our structural studies on human Dicer in complex with siRNA having different overhang lengths at 5’- and 3’-ends along with in vitro and in vivo functional studies showed that Dicer recognizes both 3’- as well as 5’-ends for proper cleavage of siRNA/miRNA. The 5'-end recognition by Dicer, demonstrated for the first time by us, is important for precise and effective biogenesis of siRNA/miRNA. This study has provided practical benefits to the design of small-RNAs for gene silencing. We also solved the crystal structure of a large complex of Trax–translin heteromers, also known as C3PO, which has been proposed to activate the RNA-induced silencing complex by facilitating endonucleolytic cleavage of the siRNA passenger strand. Our studies establish that Trax adopts the translin fold, possesses catalytic centers essential for C3PO's endoribonuclease activity and interacts extensively with translin to form an octameric assembly. This study provides important insights into the catalytic mechanism of C3PO and its conserved role in human RISC activation. Just as miRNAs and siRNAs bind to the Argonaute proteins, another class of small RNAs encoded in the genome, the Piwi-interacting RNAs (piRNAs), that are 2′-O-methylated at their 3′ ends, bind to the Piwi proteins. Germline-specific piRNAs and Piwi proteins play a critical role in genome defense against transposable elements. Our work on Piwi proteins demonstrated the structural basis for piRNA 2'-O-methylated 3' end recognition by the PAZ domain of Piwi proteins.
April 10, 2014 at 4.00 pm in AG-80
Structural Insights into Non-vesicular Trafficking of Lipids by Lipid Transfer Proteins and Bacterial Toxin-Antitoxin Systems
Phosphorylated sphingolipid Ceramide-1-Phosphate (C1P) is a key regulator of cell growth, survival, migration and inflammatory responses, but non-vesicular mechanisms involved in its intracellular sensing, transfer and presentation remained unexplored till recently. We have identified a widely-expressed protein, CPTP (Ceramide-1-Phosphate Transfer Protein) in humans, which specifically transfers C1P. Our co-crystal structures established that C1P binding occurs via a surface-localized phosphate head-group recognition center connected to a hydrophobic pocket. Down-regulation of CPTP dramatically alters C1P steady-state levels, decreasing at the plasma membrane while elevating at the trans-Golgi where C1P is produced by ceramide kinase. The elevated C1P triggers proinflammatory eicosanoid generation associated with cellular inflammation, thus highlighting its physiological importance. We have also carried out structure-function studies on lipid transfer proteins involved in cell survival such as “accelerated cell death 11 (ACD11)” protein from Arabidopsis and “heterokaryon incompatibility C2 protein (HET-C2)” from the fungus Podospora anserina which has provided insights on roles of lipid transfer process in cell survival.
Almost all bacteria and many archaea contain genes (encoding toxins) whose expression inhibit cell growth and may lead to cell death when overproduced, reminiscent of apoptotic genes in eukaryotes. These toxins are co-expressed and neutralized with their cognate antitoxins from TA (toxin-antitoxin) operons in normally growing cells. MazF (toxin) / MazE (antitoxin) system is one of the most extensively characterized TA systems. Under stress conditions, labile anti-toxins (MazE) are readily degraded by proteases allowing MazF (toxin) to cleave mRNA in a sequence specific manner. Recently, we solved the structure of Bacillus MazF in complex with ssRNA containing its target site as well as in complex with its cognate antitoxin MazE. This study has provided for the first time structural basis of recognition and cleavage of mRNA by MazF in a sequence specific manner during stress conditions and also demonstrated how antitoxin inactivates the toxin in normally growing cells.
April 7, 2014 at 4.00 pm in AG-69
Spectroscopic Studies of Interactions of Some Biologically Important Small Molecules with Proteins
Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate virtually in every process within cells. The work is based on studies of the interactions of erythroid membrane skeletal protein, Spectrin and Hb along with serum protein from human (HSA) and from bovine (BSA) and another soluble protein of non-erythroid origin, the lysozyme (Lyz) with small organic molecules like the phosphate metabolite ATP, hemin and other small molecules of therapeutic importance e.g. 4-Nitroquinoline (4NQO), Merocyanine 540 (MC 540) and Imatinib mesylate. To study these interactions we have been using spectroscopic tools like absorption, fluorescence, circular dichroism, synchronous fluorescence, time-resolved fluorescence and theoretical modelling.
SAs are the most important studied proteins in this field but the structural comparison between HSA and BSA was not studied previously. This aspect has been highlighted in this thesis while discussing the interactions of MC 540 with both HSA and BSA. The discrepancies between the spectroscopic data obtained with HSA and BSA while interacting with MC 540 depict that although both the SAs have 80% structural similarities but the conformational flexibility is greater in HSA than that of BSA that has further been confirmed by the results obtained from time-resolved fluorescence and induced CD experiments.
The Hb is the most studied blood protein and its structural changes have strong impact on blood-related or hematological diseases. In case of thalassemia, the prosthetic heme group of Hb is sometimes displaced from heme pocket. This type of phenomenon of heme loss could be stimulated in acidic pH. In erythrocyte cells, the membrane contains different types of phospholipids and cholesterol. Therefore, a detailed discussion on the impact of phospholipids on Hb has been done in my Ph. D. work. The heme loss and the release of oxygen from Hb occur simultaneously. The oxygen affinity of Hb decreases, in some diseased conditions as well as due to interaction with ATP. This aspect has been studied in detail by the spectroscopic techniques. Besides Hb, the Spectrin is also an erythrocyte membrane skeletal protein. The interaction of Spectrin with different phospholipids and drugs were studied previously. Here we have reported our findings regarding the interactions between Spectrin and an antileukemic drug, Imatinib as well as between Spectrin and another antileukemic as well as good fluorescent molecule, MC 540.
April 28, 2014 at 4.00 pm in AG-69
Elastic Network Model in describing equilibrium properties of proteins
In this talk, I will describe why elastic network model (ENM) is a faster alternative to standard molecular dynamics (MD) simulations in understanding the dynamics of proteins under equilibrium conditions. I will discuss the approaches taken for optimal parametrization of the model and how the parameters are used to study intrinsic flexibility of proteins. I will also talk about some future applications of ENM in studying specific functional motions of proteins.
March 3, 2014 at 4.00 pm in AG-69
Photoinduced Electron Transfer in DNA Repair: A Computational Study
UV radiation (200-400 nm) causes damage of DNA, mostly producing the cyclobutane
pyrimidine and (6-4)-pyrimidine-pyrimidone (PP) photodimers. (6-4) photolyase is a DNA repair enzyme that selectively repairs (6-4)PP photodimer, using visible light. This repairprocess is a complex photocycle comprising of several electron transfer (ET) steps possibly coupled to proton transfer, controlled by the protein. The key repair steps are the forward electron transfer (FET) and the back electron transfer (BET) which involve the FADH−chromophore and the (6-4)PP. Experimentally, the ET rates of these steps have been estimated, but the overall repair mechanism remains elusive.
We wish to predict the ET rates with in the photolyase repair site and evaluate the roles of various factors that may influence these ET rates. Towards this, we have set up a multiscale computational apparatus based on a combination of QM, hybrid QM/MM and MD methodologies. With this approach, we have computed the energies of the FADH− - (6-4) PP (Donor-Acceptor) complex in different electronic states, formally describing the electron transfer reaction. We then evaluate the effects of the protonation state of key residues, the solvent and DNA counterions on these states and also account for the effect of the protein dynamics. Using these complex energy estimates, we predict the FET and BET rates employing the semi classical Marcus formalism and compare them against the experimental observations.