The sequential mechanism of guanidine hydrochloride-induced denaturation of cAMP receptor protein from Escherichia coli. A fluorescent study using 8-anilino-1-naphthalenesulfonic acid.
cAMP receptor protein (CRP) regulates expression of a number of genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two structural domains. The biological activation of CRP upon cAMP binding may involve the subunit realignment as well as reorientation between the domains within each subunit. In order to study the interactions between the subunits or domains, we performed stopped-flow measurements of the guanidine hydrochloride (GuHCI)-induced denaturation of CRP. The changes in CRP structure induced by GuHCl were monitored using both intrinsic Trp fluorescence as well as the fluorescence of an extrinsic probe, 8-anilino-1-Naphthalenesulfonic acid (ANS). Results of CRP denaturation using Trp fluorescence detection are consistent with a two-step model [Malecki, and Wasylewski, (1997), Eur. J. Biochem. 243, 660], where the dissociation of dimer into subunits is followed by the monomer unfolding. The denaturation of CRP monitored by ANS fluorescence reveals the existence of two additional processes. One occurs before the dissociation of CRP into subunits, whereas the second takes place after the dissociation, but prior to proper subunit unfolding. These additional processes suggest that CRP denaturation is described by a more complicated mechanism than a simple three-state equilibrium and may involve additional changes in both inter- and intrasubunit interactions. We also report the effect of cAMP on the kinetics of CRP subunit unfolding and refolding.
Malecki
J
J
Department of Physical Biochemistry, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland.
Wasylewski
Z
Z
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011953:Receptors, Cyclic AMP / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*