https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/66 Mon, 27 Apr 2026 15:57:57 GMT 2026-04-27T15:57:57Z https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1369 Title: Asymmetric DNA methylation by dimeric EcoP15I DNA methyltransferase Authors: Roy, Rajendra P Abstract: EcoP15I DNA methyltransferase (M.EcoP15I) recognizes short asymmetric sequence, 5'-CAGCAG-3', and methylates the second adenine only on one strand of the double-stranded DNA (dsDNA). In vivo, this methylation is sufficient to protect the host DNA from cleavage by the cognate restriction endonuclease, R.EcoP15I, because of the stringent cleavage specificity requirements. Biochemical and structural characterization support the notion that purified M.EcoP15I exists and functions as dimer. However, the exact role of dimerization in M.EcoP15I reaction mechanism remains elusive. Here we engineered M.EcoP15I to a stable monomeric form and studied the role of dimerization in enzyme catalyzed methylation reaction. While the monomeric form binds single-stranded DNA (ssDNA) containing the recognition sequence it is unable to methylate it. Further we show that, while the monomeric form has AdoMet binding and Mg(2+) binding motifs intact, optimal dsDNA binding required for methylation is dependent on dimerization. Together, our biochemical data supports a unique subunit organization for M.EcoP15I to catalyze the methylation reaction. Fri, 01 Jan 2016 00:00:00 GMT https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1369 2016-01-01T00:00:00Z https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1194 Title: Sortase-click strategy for defined protein conjugation on a heptavalent cyclodextrin scaffold Authors: Roy, Rajendra P; Singh, Shikha; Gupta, Kanchan; Shukla, Shagun; Sampathkumar, Srinivasa-Gopalan Abstract: Multivalent proteins or protein dendrimers are useful for clinical and biotechnological applications. However, assembly of chemically defined protein dendrimers is a challenging endeavor. In the past, majority of protein dendrimers have been developed on branched lysine scaffolds and are usually limited to a valency of two to four. The naturally occurring cyclodextrin (CD) scaffold composed of 6-8 glucose units offers the possibility of expanding the valency. Here we have adapted a chemoenzymatic-click strategy for displaying heptavalent peptides and large proteins on the β-cyclodextrin (β-CD) scaffold. We demonstrate that recombinant proteins (engineered with a LPXTG pentapeptide motif at the carboxy terminus), labeled with an alkyne moiety by sortase-mediated ligation, can be easily clicked on to the azide-derivatized β-cyclodextrin through the Huisgen cycloaddition reaction yielding a well-defined heptavalent display of proteins. Tue, 01 Jan 2019 00:00:00 GMT https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1194 2019-01-01T00:00:00Z