Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride.
The aim of this work was to study the conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnHCl and the effect of the binding of glutamine (Gln) on these processes. To this end, GdnHCl-induced unfolding of GlnBP alone and its GlnBP-Gln complex was studied by protein intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy. The obtained spectroscopic data were interpreted taking into the account the peculiarities of protein three-dimensional structure. In particular, the fact that formation of a complex of GlnBP and Gln, which essentially changes the global structure of protein, affects only insignificantly the microenvironments of tryptophan residues elucidates the similarity of the emission spectra of GlnBP and the GlnBP-Gln complex, and the existence of quenching groups near tyrosine residues and an effective nonradiative Tyr --> Trp and/or Tyr --> Tyr --> Trp energy transfer provide an explanation for the negligibly small contribution of tyrosine to the bulk fluorescence of the native protein and for its increase in protein unfolding. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gln unfolding are three-step processes (N --> I(1) --> I(2) --> U), though in the case of the GlnBP-Gln complex these stages essentially overlap. Despite the complex character, GlnBP unfolding is completely reversible. The dramatic shift of the N --> I(1) process to higher GdnHCl concentrations for the GlnBP-Gln complex in comparison with GlnBP was shown.
Staiano
Maria
M
Institute of Protein Biochemistry, CNR, Naples, Italy.
Scognamiglio
Viviana
V
Rossi
Mose'
M
D'Auria
Sabato
S
Stepanenko
Olga V
OV
Kuznetsova
Irina M
IM
Turoverov
Konstantin K
KK
D000817:Anilino Naphthalenesulfonates; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005973:Glutamine / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D046911:Macromolecular Substances; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; C037853:glutamine transport proteins; D005973:Glutamine; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D019791:Guanidine