Characterization of a urea induced molten globule intermediate state of glutaminyl-tRNA synthetase from Escherichia coli.
The urea-induced unfolding of glutaminyl-tRNA synthetase, a multidomain protein, has been studied by equilibrium and kinetic methods, using chemical modification, fluorescence, and CD spectroscopy. The far-UV CD, fluorescence, and sulfhydryl reactivity clearly demonstrated the existence of a stable intermediate state at around 2 M urea. The intermediate showed higher binding of 1-anilino-8-naphthalenesulfonic acid. Furthermore, near-UV CD study of the intermediate showed significantly disrupted tertiary structure with only a small change in the secondary structure, which is a characteristic of molten globule states. The activation energies (delta G++) calculated from unfolding kinetics monitored by CD and fluorescence suggest that the intermediate state may be separated from the native and the unfolded state by high activation energy barriers.
Das
B K
BK
Department of Biophysics, Bose Institute, Calcutta, India.
Bhattacharyya
T
T
Roy
S
S
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds / Q000032:analysis; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
D013438:Sulfhydryl Compounds; D014508:Urea; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase