FCCC LOGO Faculty Publications
Garcia P , Bruix M , Rico M , Ciofi-Baffoni S , Banci L , Ramachandra Shastry MC , Roder H , De Lumley Woodyear T , Johnson CM , Fersht AR , Barker PD
Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562
Journal of Molecular Biology. 2005 ;346(1) :331-344
Back to previous list
Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-?-helical bundle. The holo protein apparently refolds with a half-life of 4 ?s in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 ?s that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins. © 2005 Published by Elsevier Ltd.
00222836 (ISSN) Cited By: 4; Export Date: 25 May 2006; Source: Scopus CODEN: JMOBA; DOI: 10.1016/j.jmb.2004.11.044 Language of Original Document: English Correspondence Address: Barker, P.D.; Department of Chemistry; University of Cambridge; Lensfield Road Cambridge, CB2 1EW, United Kingdom; email: pdb30@cam.ac.uk Chemicals/CAS: cytochrome b562, 9064-79-3; guanidine hydrochloride, 50-01-1; heme, 14875-96-8, Apoproteins; Cytochrome b Group; cytochrome b562, E coli, 9064-79-3; Escherichia coli Proteins; Guanidine, 113-00-8; Heme, 14875-96-8; Tryptophan, 73-22-3; Urea, 57-13-6 References: Wittung-Stafshede, P., Role of cofactors in protein folding (2002) Accts Chem. Res., 35, pp. 201-208; Baker, D., A surprising simplicity to protein folding (2000) Nature, 405, pp. 39-42; Plaxco, K.W., Simons, K.T., Ruczinski, I., Baker, D., Topology, stability, sequence, and length: Defining the determinants of two-state protein folding kinetics (2000) Biochemistry, 39, pp. 11177-11183; Stellwagen, E., Rysavy, R., Babul, G., The conformation of horse heart apocytochrome c (1972) J. Biol. Chem., 247, pp. 8074-8077; Barker, P.D., Ferguson, S.J., Still a puzzle: Why is haem covalently attached in c-type cytochromes? (1999) Structure, 7, pp. 281-R290; Hargrove, M.S., Krzywda, S., Wilkinson, A.J., Dou, Y., Ikeda-Saito, M., Olson, J.S., Stability of myoglobin: A model for the folding of heme proteins (1994) Biochemistry, 33, pp. 11767-11775; Feng, Y.Q., Sligar, S.G., Effect of heme binding on the structure and stability of Escherichia coli apocytochrome b562 (1991) Biochemistry, 30, pp. 10150-10155; Manyusa, S., Whitford, D., Defining folding and unfolding reactions of apocytochrome b(5) using equilibrium and kinetic fluorescence measurements (1999) Biochemistry, 38, pp. 9533-9540; Arnesano, F., Banci, L., Bertini, I., Faronoe-Mennella, J., Rosato, A., Barker, P.D., Fersht, A.R., The solution structure of oxidized Escherichia coli cytochrome b 562 (1999) Biochemistry, 38, pp. 8657-8670; Hamada, K., Bethge, P.H., Mathews, F.S., Refined structure of cytochrome b562 from Escherichia coli at 1.4 A? resolution (1995) J. Mol. Biol., 247, pp. 947-962; Feng, Y., Sligar, S.G., Wand, A.J., Solution structure of apocytochrome b562 (1994) Nature Struct. Biol., 1, pp. 30-34; Laidig, K.E., Daggett, V., Molecular dynamics simulations of apocytochrome b562 - The highly ordered limit of molten globules (1996) Fold. Des., 1, pp. 335-346; Assfalg, M., Banci, L., Bertini, I., Ciofi-Baffoni, S., Barker, P.D., (15)N backbone dynamics of ferricytochrome b(562): Comparison with the reduced protein and the R98C variant (2001) Biochemistry, 40, pp. 12761-12771; D'Amelio, N., Bonvin, A., Czisch, M., Barker, P.D., Kaptein, R., The C terminus of apocytocrome b562 undergoes fast motions and slow exchange among ordered conformations resembling the folded state (2002) Biochemistry, 41, pp. 5505-5514; Barker, P.D., Nerou, E.P., Freund, S.M.V., Fearnley, I.M., Conversion of cytochrome b562 to c-type cytochromes (1995) Biochemistry, 34, pp. 15191-15203; Wittung-Stafshede, P., Gray, H.B., Winkler, J.R., Rapid formation of a four-helix bundle. Cytochrome b(562) folding triggered by electron transfer (1997) J. Am. Chem. Soc., 119, pp. 9562-9563; Wittung-Stafshede, P., Lee, J.C., Gray, H.B., Winkler, J.R., Cytochrome b562 folding triggered by electron transfer: Approaching the speed limit for formation of a four-helix-bundle protein (1999) Proc. Natl Acad. Sci. USA, 96, pp. 6587-6590; Eaton, W.A., Munoz, V., Thompson, P.A., Henry, E.R., Hofrichter, J., Kinetics and dynamics of loops, alpha-helices, beta-hairpins, and fast-folding proteins (1998) Accts Chem. Res., 31, pp. 745-753; Hagen, S.J., Hofrichter, J., Szabo, A., Eaton, W.A., Diffusion-limited contact formation in unfolded cytochrome c: Estimating the maximum rate of protein folding (1996) Proc. Natl Acad. Sci. USA, 93, pp. 11615-11617; Thirumalai, D., Time scales for the formation of the most probable tertiary contacts in proteins with applications to cytochrome c (1999) J. Phys. Chem. B, 103, pp. 608-610; Telford, J.R., Wittungstafshede, P., Gray, H.B., Winkler, J.R., Protein folding triggered by electron transfer (1998) Accts Chem. Res., 31, pp. 755-763; Wittung-Stafshede, P., Effect of redox state on unfolding energetics of heme proteins (1999) Biochim. Biophys. Acta, 1432, pp. 401-405; Lee, J.C., Gray, H.B., Winkler, J.R., Cytochrome c? folding triggered by electron transfer: Fast and slow formation of four-helix bundles (2001) Proc. Natl Acad. Sci. USA, 98, pp. 7760-7764; Moore, G.R., Pettigrew, G.W., Cytochromes c: Evolutionary, Structural and Physiochemical Aspects (1990) Springer Series in Molecular Biology, Springer-Verlag Berlin; Springs, S.L., Bass, S.E., McLendon, G.L., Cytochrome b562 variants: A library for examining redox potential evolution (2000) Biochemistry, 39, pp. 6075-6082; Fersht, A.R., (1999) Structure and Mechanism in Protein Science, W.H. Freeman New York; Matouschek, A., Kellis Jr., J.T., Serrano, L., Bycroft, M., Fersht, A.R., Transient folding intermediates characterized by protein engineering (1990) Nature, 346, pp. 440-445; Shastry, M.C., Roder, H., Evidence for barrier-limited protein folding kinetics on the microsecond time scale (1998) Nature Struct. Biol., 5, pp. 385-392; Shastry, M.C.R., Sauder, J.M., Roder, H., Kinetic and structural analysis of submillisecond folding events in cytochrome c (1998) Accts Chem. Res., 31, pp. 717-725; White, A., Effect of pH on fluorescence of tyrosine, tryptophan and related compounds (1959) Biochem. J., 71, pp. 217-220; Arnesano, F., Banci, L., Bertini, I., Ciofi-Banoffi, S., De Lumley Woodyear, T., Johnson, C.M., Barker, P.D., Structural consequences of b- to c-type cytochrome conversion in oxidized Escherichia coli cytochrome b562 (2000) Biochemistry, 39, pp. 1499-1514; Van Nuland, N.A., Meijberg, W., Warner, J., Forge, V., Scheek, R.M., Robillard, G.T., Dobson, C.M., Slow cooperative folding of a small globular protein HPr (1998) Biochemistry, 37, pp. 622-637; Arnesano, F., Banci, L., Bertini, I., Koulougliotis, D., Solution structure of oxidized rat microsomal cytochrome b5 in the presence of 2 M guanidinium chloride: Monitoring the early steps in protein unfolding (1998) Biochemistry, 37, pp. 17082-17092; Arnesano, F., Banci, L., Bertini, I., Koulougliotis, D., Monti, A., Monitoring mobility in the early steps of unfolding: The case of oxidized cytochrome b(5) in the presence of 2 M guanidinium chloride (2000) Biochemistry, 39, pp. 7117-7130; Mayor, U., Johnson, C.M., Daggett, V., Fersht, A.R., Protein folding and unfolding in microseconds to nanoseconds by experiment and simulation (2000) Proc. Natl Acad. Sci. USA, 97, pp. 13518-13522; Barker, P.D., Nerou, E.P., Cheesman, M.R., Thomson, A.J., De Oliveira, P., Hill, H.A.O., Bis-methionine ligation to heme iron in mutants of cytochrome b 562. 1. Spectroscopic and electrochemical characterisation of the electronic properties (1996) Biochemistry, 35, pp. 13618-13626; Barker, P.D., Freund, S.M.V., Bis-methionine ligation to heme iron in mutants of cytochrome b 562. 2. Characterization by NMR of heme-ligand interactions (1996) Biochemistry, 35, pp. 13627-13635; Arcovito, A., Gianni, S., Brunori, M., Travaglini-Allocatelli, C., Bellelli, A., Fast coordination changes in cytochrome c do not necessarily imply folding (2001) J. Biol. Chem., 276, pp. 41073-41078; Robinson, C.R., Liu, Y.F., O'Brien, R., Sligar, S.G., Sturtevant, J.M., A differential scanning calorimetric study of the thermal unfolding of apo- and holo-cytochrome b562 (1998) Protein Sci., 7, pp. 961-965; Robinson, C.R., Liu, Y.F., Thomson, J.A., Sturtevant, J.M., Sligar, S.G., Energetics of heme binding to native and denatured states of cytochrome b562 (1997) Biochemistry, 36, pp. 16141-16146; Dinner, A.R., Karplus, M., The roles of stability and contact order in determining protein folding rates (2001) Nature Struct. Biol., 8, pp. 21-22; Fersht, A.R., Transition-state structure as a unifying basis in protein- folding mechanisms: Contact order, chain topology, stability, and the extended nucleus mechanism (2000) Proc. Natl Acad. Sci. USA, 97, pp. 1525-1529; Gill, S.C., V Hippel, P.H., Calculation of protein extinction coefficients from amino acid sequence data (1989) Anal. Biochem., 182, pp. 319-326; Shastry, M.C., Luck, S.D., Roder, H., A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale (1998) Biophys. J., 74, pp. 2714-2721; Wider, G., Neri, D., Otting, G., Wuthrich, K., A heteronuclear 3-dimensional NMR experiment for measurements of small heteronuclear coupling-constants in biological macromolecules (1989) J. Magn. Reson., 85, pp. 426-431; Vuister, G.W., Bax, A., Quantitative J correlation - A new approach for measuring homonuclear 3-bond J(H(N)H(alpha) coupling-constants in N-15- enriched proteins (1993) J. Am. Chem. Soc., 115, pp. 7772-7777; Johnson, B.A., Blevins, R.A., NMR View-a computer program for the visualization and analysis of NMR data (1994) J. Biomol. NMR, 4, pp. 603-614; MacUra, S., Wuthrich, K., Ernst, R.R., The relevance of J-cross-peaks in two-dimensional NOE experiments of macromolecules (1982) J. Magn. Reson., 47, pp. 351-357; Marion, D., Wuthrich, K., Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins (1983) Biochem. Biophys. Res. Commun., 113, pp. 967-974; Farrow, N.A., Zhang, O., Forman-Kay, J.D., Kay, L.E., A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium (1994) J. Biomol. NMR, 4, pp. 727-734; Mulder, F.A., Van Tilborg, P.J., Kaptein, R., Boelens, R., Microsecond time scale dynamics in the RXR DNA-binding domain from a combination of spin-echo and off-resonance rotating frame relaxation measurements (1999) J. Biomol. NMR., 13, pp. 275-288.