[The structure and stability of the glutamine-binding protein from Escherichia coli and its complex with glutamine].
A study was made of the conformational changes in the Escherichia coli glutamine-binding potein (GlnBP) induced by GdnHCl, and of the effect of glutamine (Gln) binding on these processes. Intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy were used. The obtained experimental data were interpreted, taking into the account results of the analysis of tryptophan and tyrosine residues microenvironments. This enabled us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of fluorescence characteristics of GlnBP and GlnBP/Gln, and an uncommon effect of the excess of fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm compared to the excitation at 280 nm. The latter effect is explained by the spectral dependence of Trp 32 and Trp 220 contributions to protein absorption. The dependence of Trp fluorescence of protein on the excitation wavelength must be taken into account for the evaluation of Tyr residues contribution to the bulk fluorescence of protein, and in principle, it may also be used for the development of an approach to decomposition of multi-component protein fluorescence spectrum. The parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP/Gln unfolding are three-step processes (N-->I1-->I2-->U), though in the case of the GlnBP/Gln complex these stages essentially overlap. Despite its complex character, GlnBP unfolding is completely reversible. In comparison with GlnBP, in the case of GlnBP/Gln the dramatic shift of N-->I1 process to higher GdHCl concentrations is shown.
Stepanenko
Ol'ga V
OV
Kuznetsova
I M
IM
Turoverov
K K
KK
Scognamiglio
V
V
Staiano
M
M
D'Auria
S
S
D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D005453:Fluorescence; D005973:Glutamine / Q000378:metabolism*; D019791:Guanidine; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013057:Spectrum Analysis; D014443:Tyrosine
D029968:Escherichia coli Proteins; D005973:Glutamine; D014443:Tyrosine; D019791:Guanidine