RESUMO
We have investigated the folding dynamics of Thermus thermophilus cytochrome c(552) by time-resolved fluorescence energy transfer between the heme and each of seven site-specific fluorescent probes. We have found both an equilibrium unfolding intermediate and a distinct refolding intermediate from kinetics studies. Depending on the protein region monitored, we observed either two-state or three-state denaturation transitions. The unfolding intermediate associated with three-state folding exhibited native contacts in ß-sheet and C-terminal helix regions. We probed the formation of a refolding intermediate by time-resolved fluorescence energy transfer between residue 110 and the heme using a continuous flow mixer. The intermediate ensemble, a heterogeneous mixture of compact and extended polypeptides, forms in a millisecond, substantially slower than the â¼100-µs formation of a burst-phase intermediate in cytochrome c. The surprising finding is that, unlike for cytochrome c, there is an observable folding intermediate, but no microsecond burst phase in the folding kinetics of the structurally related thermostable protein.
Assuntos
Proteínas de Bactérias/química , Grupo dos Citocromos c/química , Heme/química , Dobramento de Proteína , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Heme/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , Mutação , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , Espectrometria de Fluorescência , Thermus thermophilus/genética , Thermus thermophilus/metabolismo , Fatores de TempoRESUMO
We have investigated intrachain contact dynamics in unfolded cytochrome cb562 by monitoring heme quenching of excited ruthenium photosensitizers covalently bound to residues along the polypeptide. Intrachain diffusion for chemically denatured proteins proceeds on the microsecond time scale with an upper limit of 0.1 µs. The rate constants exhibit a power-law dependence on the number of peptide bonds between the heme and Ru complex. The power-law exponent of -1.5 is consistent with theoretical models for freely jointed Gaussian chains, but its magnitude is smaller than that reported for several synthetic polypeptides. Contact formation within a stable loop was examined in a His63-heme ligated form of the protein under denaturing conditions. Loop formation accelerated contact kinetics for the Ru66 labeling site, owing to reduction in the length of the peptide separating redox sites. For other labeling sites within the stable loop, quenching rates were modestly reduced compared to the open chain polymer.