Ragunathan Devendran

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9873646140

Martin-Luther University

Section Microbial Biotechnology, Institute for Biochemistry and Biotechnology, Martin luther University, Germany

The Class IVa Glycine Rich RNA binding proteins are the sub group of proteins within family of proteins called Glycine Rich Proteins (GRP). GRPs are characterized by the presence high content of amino acid glycine.   The Class IVa GRPs were reported to perform plethora of biological activities such as circadian rhythm, regulation of hormones, biotic and abiotic stress adaptation. Structurally Class IVa has two domains; structured N-terminal containing RNA Recognition Motif (RRM) and flexible C-terminal with numerous glycine making the part intrinsically disordered. Unpublished observations show that Glycine rich RNA binding protein interacted with satellite conserved region of Radish Leaf curl virus Beta satellite which belongs to Geminivirus by yeast monohybrid screen. Geminiviruses are single stranded DNA viruses that are known to infect economically important plants from the family Solanaceae. To validate the interaction a GRP from N. benthamiana named as NbGRP3 cloned into pET SUMO vector, expressed and purified. The monohybrid results were validated by EMSA and intrinsic fluorescence. Apart from this biochemical and biophysical characterisations were also done. Nucleic acid binding studies with homodeoxyribonucleotide polymers indicated that NbGRP3 bound to pyrimidines and not to purines. Analytical Ultra Centrifugation experiments showed that NbGRP3 exists as monomer in absence of nucleic acids, but when NbGRP3 was titrated with homodeoxycytosine (dC12) there were two protein molecules bound to single molecule of dC12 when the concentration of dC12 and protein were at a ratio of 1:2 (DNA:Protein), surprisingly when the concentration of dC12 increased further there was a shift from two protein molecule to single protein molecule per dC12.  This observation shows that NbGRP3 exhibit unusual mode of binding with nucleic acids. In an attempt to identify the regions that could possibly be responsible for this behaviour we ended up in identifying repeats of amino acids. Upon further investigation it was found that such repeats of similar amino acids are present in Class IVa GRP from other species such as Arabidopsis, wheat and barley. In order to understand their role in mode of nucleic acid binding, repeats were systematically deleted by PCR and cloning which resulted in 3 variants. The future work will be to express, purify the variants. It will also be interesting be see the impact of the deletions in the overall structure of the protein.

Continuing my PhD in host institution