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 Es cherichia 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 do mains within each subunit. In order to study the interactions between the s ubunits or domains, we performed stopped-flow measurements of the guanidine hydrochloride (GuHCl)-induced denaturation of CRP. The changes in CRP stru cture induced by GuHCl were monitored using both intrinsic Trp fluorescence as well as the fluorescence of an extrinsic probe, 8-anilino-1-Naphthalene sulfonic acid (ANS). Results of CRP denaturation using Trp fluorescence det ection are consistent with a two-step model [Malecki, and Wasylewski, (1997 ), Eur. J. Biochem. 243, 660], where the dissociation of dimer into subunit s is followed by the monomer unfolding. The denaturation of CRP monitored b y ANS fluorescence reveals the existence of two additional processes. One o ccurs before the dissociation of CRP into subunits, whereas the second take s place after the dissociation, but prior to proper subunit unfolding. Thes e additional processes suggest that CRP denaturation is described by a more complicated mechanism than a simple three-state equilibrium and may involv e additional changes in both inter- and intrasubunit interactions. We also report the effect of cAMP on the kinetics of CRP subunit unfolding and refo lding.