Investigation of the interaction between isolated C-module of tyrosyl-tRNA synthetase and tRNA by fluorescence spectroscopy
DOI:
https://doi.org/10.7124/bc.00073AKeywords:
tyrosyl-tRNA synthetase, mutagenesis, fluorescenceAbstract
The non-catalytic COOH-terminal module of mammalian tyrosyl-tRNA synthetase manifests a dual function: involvement in tRNA binding as a cis-factor and cytokine activity after proteolytic cleavage from synthetase catalytic core similar to EMAP II cytokine. The C-module of TyrRS contains a single Trp144 which is an intrinsic fluorescent probe in protein structure, but it is localized outside of RNA binding site. To explore the interaction between C-module and tRNA in solution the conservative aromatic Phe127 residue was substituted for Trp127 fluorophore by site-directed mutagenesis. This replacement allowed enhancing the protein quantum yield and determining the binding parameters of tRNA and C-module. The dissociation constant of the complex between C-module and tRNAPhe was calculated on the basis of spectrofluorometric titrations data. It was about 2.9·10–8M, and stoichiometry of the complex n=1.2.References
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