Dynamics of Biological Macromolecules Investigated by Fluorescence Depolarization.
The fast dynamics of biomolecular systems are believed to be important to their physiological function. Fluorescence depolarization is a powerful technique for the investigation of these dynamics. A general overview of the use of time -resolved fluorescence to investigate nanosecond dynamics is presented. The basic theory and experimental techniques for measuring time-resolved fluorescence properties in the frequency domain are outlined. Data obtained in fluorescence depolarization experiments is highly complex. Mathematical models for analyzing data from depolarization due to rotational motion have been largely based on the diffusion equation. It has been implicitly stated that a "jump" model should give the same result for the anisotropy decay as the diffusion equation. In this work, we derive the general result from the jump model, where the excitation and emission dipoles are not necessarily coincident with any of the principal rotational axes of the fluorophore. This result is found to be different from that of the diffusion equation. This difference is significant since, for systems where the fluorophore is not much larger than the solvent molecules or where the molecule may be limited to a few preferred orientations (for example, residues in proteins), the actual physical mechanism of rotation may not be accurately represented by continuous diffusion. Since there are cases where symmetry causes the two models to agree, it is proposed that both models are only limiting cases of the underlying physical process of rotational motion. The physical assumptions behind the two models and the limits of applicability of each approach are discussed, and some of the thermodynamic properties are considered. Finally, several applications of the compartmental jump approach are presented: a free disk-like molecule, a rod-like molecule in a phospholipid bilayer, and a tryptophan residue in a protein matrix.
- Pub Date:
- Biophysics: General; Physics: Molecular