title: Nanosecond-regime correlation time scales for equilibrium protein structural fluctuations of metal-free cytochrome c from picosecond time-resolved fluorescence spectroscopy and the dynamic Stokes shift.
abstract: We used picosecond time-resolved fluorescence spectroscopy to characterize the fluorescence Stokes shift (FSS) response function of metal-free (or free-base, fbCytc) cytochrome c under the solution conditions that favor the native states of ferricytochrome c (FeCytc) and Zn(II)-substituted cytochrome c (ZnCytc). The intrinsic porphyrin chromophore serves in these experiments as a fluorescent probe of the structural fluctuations of the surrounding protein and solvent. Demetalation of the porphyrin destabilizes the folded structure of cytochrome c owing to the loss of the axial metal-histidine and metal-methionine bonds. Thus, these experiments examine how the time scales detected in a dynamic solvation experiment in a chromoprotein report changes in the character of motion. The FSS response function in fbCytc in water and pH 7 is well described by a biexponential response over the 100 ps to 50 ns regime with time constants of 1.4 and 9.1 ns; under similar conditions, ZnCytc exhibits a biexponential FSS response with time constants of 250 ps and 1.5 ns [Lampa-Pastirk and Beck, J. Phys. Chem. B 2004, 108, 16288]. These time constants correspond, respectively, to the correlation time scales for motions of the hydrophobic core and the solvent-contact layer of the protein. Both of the time constants observed in fbCytc are further lengthened upon addition of glycerol to the external solvent so that a significant fraction of the protein dynamics is rendered effectively static on the fluorescence time scale. The solvation reorganization energy, the time-integrated Stokes shift of the fluorescence spectrum, is reduced by about a third to 33 cm(-1) in 50% glycerol from 43 cm(-1) in water. These results are interpreted structurally using a model for Brownian diffusive motion with thermally activated barrier crossings on the protein-folding energy landscape. The results suggest that the mean-squared deviations of the structural fluctuations exhibited by fbCytc are nearly a factor of 10 larger than those of ZnCytc. This conclusion is consistent with the suggestion that fbCytc assumes a dynamic, partially unfolded structure with some of the characteristics of a molten globule.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Jagnyaseni
authors.initials: J
authors.lastname: Tripathy
authors.forename: Warren F
authors.initials: WF
authors.lastname: Beck
mesh_terms: D045304:Cytochromes c / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors; D014867:Water / Q000737:chemistry
chemical_list: D014867:Water; D045304:Cytochromes c