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Doctoral Research

Role RNA-protein complexes in regulating host-pathogen interactions

Non-coding RNAs and their protein (RNP) complexes consisting of both viral and human components contribute significantly to the regulation of viral pathogenesis. Thus, I aim to understand the role of native human-HIV RNP complexes in regulation of virus replication and latency to generate novel therapeutic strategies. Towards this aim, I am using an integrated structural biology approach consisting of Cryo-Electron Microscopy and X-ray Crystallography to determine high-resolution structures of key complexes regulating transcriptional activation and translational initiation of HIV mRNA. Next, I will study the role of structural dynamics in assembly and function of such complexes to determine the hotspots of host-pathogen interactions for development of anti-HIV strategies. These studies are the first steps towards constructing a comprehensive, dynamic and high-resolution structure-function interactome of RNP complexes involved in infectious diseases to open novel avenues for aiding global health security efforts.

Bringing functional order to disordered complexes mediating human-HIV interactions

HIV hijacks the human transcription machinery to make multiple copies of its own genome. This process, known as transactivation (TAC), is crucial in the HIV infection cycle and has thus become the object of focused scientific attention in the past two decades - both for understanding its molecular mechanism and for the development of anti-HIV drugs. Numerous studies have revealed that the viral components of TAC – TAR RNA and Tat protein – are highly flexible and intrinsically disordered molecules respectively and undergo significant conformational changes while assembling into multipartite complexes. Such properties have impeded the formation of comprehensive and homogenous TAC complexes for high-resolution structural biology analyses. I have addressed this challenge by systematically probing the construction of stable TAC ribonucleoprotein complexes [1] in solution and am currently using this method for determining the structure and dynamics of the whole HIV-1 TAC by X-ray crystallography complemented by NMR spectroscopy.

Determination of the structure of low-populated intermediates in RNA-protein recognition

All biochemical reactions in living organisms require molecular recognition events. In particular, the interactions between protein and RNA molecules are crucial in the regulation of gene expression. However, the transient nature of the conformations populated during the recognition process has prevented a detailed characterization of the mechanisms by which these interactions take place. To address this problem, we report a high-resolution structure of an intermediate state in protein-RNA recognition. We determined this structure by using NMR measurements as ensemble-averaged structural restraints in metadynamics simulations, and validated it by performing a structure-based design of two mutants with rationally modified binding rates.

Constructing free energy landscapes of RNA

RNA molecules in solution tend to undergo structural fluctuations of relatively large amplitude and to populate a range of different conformations some of which with low populations. It is still very challenging, however, to characterise the structures of these low populated states and to understand their functional roles. In the present study, we address this problem by using NMR residual dipolar couplings (RDCs) as structural restraints in replica-averaged metadynamics (RAM) simulations. By applying this approach to a 14-mer RNA hairpin containing the prototypical UUCG tetraloop motif, we show that it is possible to construct the free energy landscape of this RNA molecule. This free energy landscapes reveals the surprisingly rich dynamics of the UUCG tetraloop and identifies the multiple substates that exist in equilibrium owing to thermal fluctuations. The approach that we present is general and can be applied to the study of the free energy landscapes of other RNA or RNA-protein systems.

Automation of NMR spectra assignments to aid high-throughput structural biology of proteins

Development of efficient strategies and automation represent important milestones of progress in rapid structure determination efforts in proteomics research. In this context, we developed an efficient algorithm named as AUTOBA (Automatic Backbone Assignment) designed to automate the assignment protocol based on HN(C)N suite of experiments. Depending upon the spectral dispersion, the user can record 2D or 3D versions of the experiments for assignment. The algorithm uses as inputs: (i) protein primary sequence and (ii) peak-lists from user defined HN(C)N suite of experiments. In the end, one gets H(N), (15)N, C(α) and C' assignments (in common BMRB format) for the individual residues along the polypeptide chain. The success of the algorithm has been demonstrated, not only with experimental spectra recorded on two small globular proteins: ubiquitin (76 aa) and M-crystallin (85 aa), but also with simulated spectra of 27 other proteins using assignment data from the BMRB.

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AUTOBA can be accessed as an online server here. Alternatively, it can be downloaded as a stand alone C code with a JAVA GUI here

Molecular modelling of DNA in torsion angle hyperspace

Analysis of the conformational space populated by the torsion angles and the correlation between the conformational energy and the sequence of DNA are important for fully understanding DNA structure and function. Presence of seven variable torsion angles about single covalent bonds in DNA main chain puts a big challenge for such analysis. We have carried out restrained energy minimization studies for four representative dinucleosides, namely d(ApA):d(TpT), d(CpG):d(CpG), d(GpC):d(GpC) and d(CpA):d(TpG) to determine the energy hyperspace of DNA in context to the values of the torsion angles and the structural properties of the DNA conformations populating the favorable regions of this energy hyperspace. The torsion angles were manipulated by constraining their values at the reference points and then performing energy minimization. The energy minima obtained on the potential energy contour plots mostly correspond to the conformations populated in crystal structures of DNA. Some novel favorable conformations that are not present in crystal structure data are also found. The plots also suggest few low energy routes for conformational transitions or the associated energy barrier heights. Analyses of base pairing and stacking possibility reveal structural changes accompanying these transitions as well as the flexibility of different base steps towards variations in different torsion angles

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Master's Research
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Postdoc Research
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