Abstract
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 (GuHCl)-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.
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REFERENCES
Aiba, H., Fujimoto, S., and Ozaki, N. (1982). Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein, Nucleic Acids Res. 10, 1345–1361.
Bernasconi, C. F. (1976). Relaxation Kinetics, Academic Press, New York.
Cheng, X., Gonzales, M. L., and Lee, J. C. (1993). Energetics of intersubunit and intrasubunit interactions of Escherichia coli adenosine cyclic 3′–5′-phosphate receptor protein, Biochemistry 32, 8130–8139.
Cheng, X., Kovac, L., and Lee, J. C. (1995). Probing the mechanism of CRP activation by site-directed mutagenesis: The role of serine 128 in the allosteric pathway of cAMP receptor protein, Biochemistry 34, 10816–10826.
de Crombrugghe, B., Busby, S., and Buc, H. (1984). Cyclic AMP receptor protein: Role in transcription activation, Science 224, 831–838.
Donoso-Pardo, J. L., Turner, P. C., and King, R. W. (1987). Cyclic nucleotide binding to cAMP receptor protein from Escherichia coli. Optical and ligand-binding studies, Eur. J. Biochem. 168, 687–694.
Fergusson, R. N., Edelhoch, H., Saroff, H. A., Robbins, J., and Cahnmann, H. J. (1975). Negative cooperativity in the binding of thyroxine to human serum prealbumin. Preparation of tritium-labeled 8-anilino-1-naphtalenesulfonic acid, Biochemistry 14, 282–289.
Gerl, M., Jaenicke, R., Smith, J. M. A., and Harrison, P. M. (1988). Self-assembly of apoferritin from horse spleen after reversible chemical modification with 2,3-dimethylmaleic anhydride, Biochemistry 27, 4089–4096.
Ghosaini, L. R., Brown, A. M., and Sturtevant, J. M. (1988). Scanning calorimetry study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides, Biochemistry 27, 5257–5261.
Gittelman, M. S., and Matthews, C. R. (1990). Folding and stability of Trp aporepresor from Escherichia coli, Biochemistry 29, 7011–7020.
Heyduk, T., and Lee, J. C. (1989). Escherichia coli cAMP receptor protein: Evidence for three protein conformational states with different promoter binding activities, Biochemistry 28, 6914–6924.
Horowitz, P. M., and Criscimagna, N. L. (1985). Differential binding of the fluorescent probe 8-anilinonaphtalene-2 sulfonic acid to rhodanese catalytic intermediates, Biochemistry 24, 2587–2593.
Jaenicke, R., (1991). Local structures, domains, subunits and assemblies, Biochemistry 30, 3147–3161.
Jaenicke, R., Rudolf, R., and Feingold, D. S. (1986). Dissociation and in vitro reconstitution of bovine liver uridine diphosphoglucose dehydrogenase. The paired subunit nature of the enzyme, Biochemistry 25, 7283–7287.
Malecki, J., and Wasylewski, Z. (1997). Stability and kinetics of unfolding and refolding of cAMP receptor protein from Escherichia coli, Eur. J. Biochem. 243, 660–669.
Malecki, J., and Wasylewski, Z. (1998). The effect of Ser 128 substitution on the structure and stability of cAMP receptor protein from Escherichia coli, J. Protein Chem., 17, 219–228.
Matthews, C. R. (1987). Effect of point mutations on the folding of globular proteins, Meth. Enzymol. 154, 498–511.
McKay, D. B., Weber, I. T., and Steitz, T. A. (1982). Structure of catabolite gene activator protein at 2.9 A resolution, J. Biol. Chem. 257, 9518–9524.
Pace, C. N. (1986). Determination and analysis of urea and guanidine hydrochloride denaturation curves, Meth. Enzymol. 131, 266–280.
Passner, J. M., and Steitz, T. A. (1997). The structure of a CAP-DNA complex having two cAMP molecules bound to each monomer, Proc. Natl. Acad. Sci. USA 94, 2843–2847.
Reznikoff, W. S. (1992). Catabolite gene activator protein activation of lac transcription, J. Bacteriol. 174, 655–658.
Ryu, S., Kim, J., Adhya, S., and Garges, S. (1993). Pivotal role of amino acid at position 138 in the allosteric hinge reorientation of cAMP receptor protein, Proc. Natl. Acad. Sci. USA 90, 75–79.
Schultz, S., Shields, G., and Steitz, T. (1991). Crystal structure of a CAP-DNA complex: The DNA is bent by 90 degrees, Science 253, 1001–1007.
Takahashi, M., Blazy, B., and Baudras, A. (1980). An equilibrium study of the cooperative binding of adenosine cyclic 3′,5′-mono-phosphate and guanosine cyclic 3′,5′-monophosphate to the adenosine cyclic 3′,5′-monophosphate receptor protein from Escherichia coli, Biochemistry 19, 5124–5130.
Uversky, V. N., Winter, S., and Lober, G. (1996). Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state, Biophys. Chem. 60, 79–88.
Wasylewski, M., Malecki, J., and Wasylewski, Z. (1995). Fluorescence study of Escherichia coli cyclic AMP receptor protein, J. Protein Chem. 14, 299–308.
Weber, I. T., and Steitz, T. A. (1987). Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution, J. Mol. Biol. 198, 311–326.
Zhang, Y. L., Zhou, J. M., and Tsou, C. L. (1996). Sequential unfolding of adenylate kinase during denaturation by guanidine hydrochloride, Biochem. Biophys. Acta 1295, 239–244.
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Malecki, J., Wasylewski, Z. The Sequential Mechanism of Guanidine Hydrochloride-Induced Denaturation of cAMP Receptor Protein from Escherichia coli. A Fluorescent Study Using 8-Anilino-1-Naphthalenesulfonic Acid. J Protein Chem 17, 745–755 (1998). https://doi.org/10.1023/A:1020718016274
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DOI: https://doi.org/10.1023/A:1020718016274