Sequential Two-Photon Delayed Fluorescence Anisotropy for Macromolecular Size Determination.

Time-resolved fluorescence anisotropy (FA) uses the fluorophore depolarization rate to report on rotational diffusion, conformation changes, and intermolecular interactions in solution. Although FA is a rapid, sensitive, and nondestructive tool for biomolecular interaction studies, the short (∼ns) fluorescence lifetime of typical dyes largely prevents the application of FA on larger macromolecular species and complexes. By using triplet shelving and recovery of optical excitation, we introduce optically activated delayed fluorescence anisotropy (OADFA) measurements using sequential two-photon excitation, effectively stretching fluorescence anisotropy measurement times from the nanosecond scale to hundreds of microseconds. We demonstrate this scheme for measuring slow depolarization processes of large macromolecular complexes, derive a quantitative rate model, and perform Monte Carlo simulations to describe the depolarization process of OADFA at the molecular level. This setup has great potential to enable future biomacromolecular and colloidal studies.

Dynamic solvation and coupling of the hydration shell of Zn(II)-substituted cytochrome c in the presence of guanidinium ions.

The fluorescence Stokes shift (FSS) response of Zn(II)-substituted cytochrome c (ZnCytc) is transformed from a monotonic red-shifting response in water to a bidirectional response with much slower time constants in the presence of low concentrations of guanidinium (Gdm(+)) ions. The FSS response in water observed over the 100 ps to 10 ns range has two exponential components with time constants of 135 ps and 1.6 ns that account for a total shift of 30 cm(-1), about one-half of the solvation reorganization energy. In contrast, in the presence of only 0.25 M Gdm(+), the FSS response initially shifts 21 cm(-1) to the blue with a 820 ps time constant and then shifts 60 cm(-1) back to the red with a 3.5 ns time constant. The effect of Gdm(+) on the FSS response effectively saturates at 1.0 M, well below the 1.75 M midpoint of the two-state unfolding transition. These results establish that the FSS response in ZnCytc includes a significant contribution from the surrounding hydration shell, which assumes a perturbed hydrogen-bonding network owing to the binding of Gdm(+) ions to the protein surface. The blue-shifting part of the FSS response arises from a light-induced conformational change that expands the protein- and solvent-derived cavity around the excited-state Zn(II) porphyrin. This non-polar part of the solvation response is enhanced in the presence of Gdm(+) because the protein/solvent surroundings of the Zn(II) porphyrin are effectively more flexible than in water. The enhanced flexibility in the presence of Gdm(+) increases the amplitudes and accordingly lengthens the correlation time scales for the protein and hydration-shell fluctuations that contribute to the FSS response.

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.

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.