Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple spectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea or guanidine-HCl followed by a reduction in intensity at intermediate denaturant concentrations; a transition at higher denaturant was detected as decreased fluorescence intensity and a red-shifted emission. Solute quenching of fluorescence indicated that tryptophans become progressively solvent-exposed during unfolding. Wild type enzyme had stronger negative CD bands between 220 and 230 nm than the mutant, indicative of greater alpha-helical content. Urea or guanidine-HCl caused a reduction in ellipticity at 222 nm at low denaturant concentration with the wild type enzyme, a transition that is absent in the mutant; both enzymes exhibited a cooperative transition at higher denaturant concentrations. Both enzymes dissociate to monomers in 1.5 m urea. Unfolding of wild type enzyme is described by a multistate unfolding and a parallel two state unfolding; the two-state component is absent in the mutant. Changes in spectral properties associated with unfolding were largely reversible after dilution to low denaturant. Unfolding of glycyl-tRNA synthetase is complex with a native state, a native-like monomer, partially unfolded states, and the unfolded state. Study holds ProTherm entries: 10519, 10520, 10521, 10522, 10523, 10524, 10525, 10526 Extra Details: Transition 1; DTT(0.05 mM) and EDTA(0.1 mM) were added in the experiment solvent-exposed; alpha-helical content; cooperative transition; multistate unfolding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:40 p.m.
|Number of data points||16|
|Assays/Quantities/Protocols||Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence MADPKIEEILAPLRANVKEQGDLVRKLKEEKAPEIDIKKAVAELKTRKKILEDKELSLAPAEDLFDRAKMEDLIKRRFFYDQSFAIYGGITGQFDFGPMGCALKSNMIHLWKKFFILQEQMLEVECSILTPEPVLKASGHVERFADLMTKDIKTGECFRLDHLIKGHLEKIKSDKNTKIELKAEIEDILIKLDGMNADEMSALMKRFEMKSPISGNDLTPPIEFNLMFNTQIGPSGLVKGFLRPETAQGIFVNFKRLLEFNQGRLPFAAAQIGNSFRNEISPRSGLLRVREFTMCEIEHFCDVKEHPKFESVKNTQSLLYSADNQEQGKPADLTTIGDAVCKGIVNNETLGYFMARIHMYMLAVGIDPKRLRFRQHMGNEMAHYACDCWDAECLSSYGWIECVGCADRSAYDLTQHTKATGIRLAAEKKLPAPKQIEVVEAIANNGRIGKAFKKDSQAINDTLATLDNAALEEMQKELDSNGEYTLITARGEFKLTPSLVNVKKTQKTIHVEEIIPSVIEPSFGVGRILYCILEHNFRMREGDEQRTYFSLPPTVAPMKCVVLPLSGNAEFQPFVRDLSQELITVDVSHKVDDSSGSIGRRYARTDELGVPYAVTVDFDTIKEPHTVTLRERDSMRQVRLPMADVPTVVRDLSNSKILWSDVEQKYPKFEQQETVKGTSV|