Determination of Rotational Correlation Times from Deconvoluted 6,7-Dimethyl-8-ribityllumazine and Lumazine Protein from Photobacterium Fluorescence Anisotropy Decay Curves. Demonstration with Zeiognathi as Fluorescent Indicatorst

The experimental and analytical protocols required far obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein ( M , 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluoresence lifetime ( 7 = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns a t room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of 7 had virtually no effect on the rotational correlation time of the lumazine protein (4 = 9.4 ns, 19 "C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (M, 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 "C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (7 = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps). Brownian rotation of fluorescent biopolymers can be directly followed via the time course of the polarized emission components after excitation with polarized light pulses. Information concerning rotational diffusiofi of macromolecules has been reviewed in the last two decades (Tao, 1969; Yguerabide, 1972; Rigler & Ehrenberg, 1973; Wahl, 1983). The method in which polarized fluorescence is created by a short pulse of polarized light is a relaxation method. It measures the return to randomization of a previously created, anisotropic distribution of fluorescent molecules. The time constant of this process (the rotational correlation time) is proportional to the size of the fluorescent particle. The time dependence of the fluorescence anisotropy is related to the fluorescence intensity polarized parallel [ II ( t ) ] and perpendicular [ I ( ? ) ] to the polarized excitation light: (1) r ( t ) = [II(t) I ( t ) l / [ l l ( f ) + 2 1 ( t ) l The experimentally measurable quantity r ( t ) is also proportional to the autocorrelation function for the dipole reorientation angle (Fleming et al., 1976): r ( t ) = 2/55(P2[e(o)-e(t)l) = 2/55[3/22(cos2 4 0 ) Y21 (2) P2 is the second Legendre polynomial of the dipole reorientation angle w a t time t , e being unit vectors along the transition dipole at t = 0 or t = t , respectively. In eq 2 we assume that absorption and emission vectors coincide. In principle, +This work was supported by the Netherlands Foundation for Chemical Research (S.O.N), with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O), National Institutes of Health Grant GM28139, and NATO Grant 835/83. *Department of Biochemistry, Agricultural University. 'Department of Molecular Physics, Agricultural University. 11 University of Georgia. the expression for ( P 2 ) is dependent on the shape of the molecule. In many examples, however, ( P 2 ) will decay according to a spherical rotor, i.e. (P2[e(O)*e(t)l) = exp(-t/@) (3) where 4 is 1/(6D), D being the rotational diffusion coefficient with D = kT/(6Vq), k is the Boltzmann constant, q is the solvent viscosity, V is the molecular volume, and T is the absolute temperature. For nonspherical molecules the time dependence of the anisotropy is more complex, and anisotropic rotation gives rise to five exponential terms for an irregularly shaped body (Belford et al., 1972). In practical cases it can be shown from numerical analysis that at most three exponential terms can be distinguished (Small & Isenberg, 1977). Ellipsoids of revolution contain three exponential terms (Tao, 1969). In practice, however, three-component anisotropy decays have never been observed. Evidence for the existence of anisotropic rotation has been provided recently from time-resolved fluorescence anisotropy measurements using three different fluorescent lipid molecules that were noncovalently attached to a specific lipid transport protein (Berkhout et al., 1984). In the three different cases the anisotropy decayed according to a single exponential decay law, but the correlation times were widely different. Apart from rotational motion of spherical and nonspherical rotors, fluorescence anisotropy decay has also been applied to investigate segmental flexibility in proteins and nucleic acids. Among the many available examples we want to mention the flexibility in an antibody molecule (Yguerabide et al., 1970), the flexibility of hinged macromolecules in general (Harvey & Cheung, 1980), and the torsional dynamics of DNA (Thomas et al., 1980). Finally, fluorescence anisotropy decay of fluorophores embedded in artificial bilayer membranes has been extensively 0006-2960/85/0424-1489$01.50/0