Fluorescence depolarization studies of filamentous actin analyzed with a genetic algorithm

Abstract

A new method, in which a genetic algorithm was combined with Brownian dynamics and Monte Carlo simulations, was developed to analyze fluorescence depolarization data collected by the time-correlated single photon-counting technique. It was applied to studies of BODIPY-labeled filamentous actin (F-actin). The technique registered the local order and reorienting motions of the fluorophores, which were covalently coupled to cysteine 374 (C374) in actin and interacted by electronic energy migration within the actin polymers. Analyses of F-actin samples composed of different fractions of labeled actin molecules revealed the known helical organization of F-actin, demonstrating the usefulness of this technique for structure determination of complex protein polymers. The distance from the filament axis to the fluorophore was found to be considerably less than expected from the proposed position of C374 at a high filament radius. In addition, polymerization experiments with BODIPY-actin suggest a 25-fold more efficient signal for filament formation than pyrene-actin.

FIGURE 1

The location of C374 targeted with the BODIPY label, and identification of the structural parameters used for assessing the position of this residue in the actin after polymerization. To the left, the molecular structure of G-actin (37,38) is displayed as a ribbon diagram with C374 indicated (balls and sticks) together with the BODIPY fluorophore. The helical structure to the right represents a hypothetical actin filament. In the center, the parameters, which specify the position of the monomers and the fluorescent group in the polymer are defined. The Z_C axis coincides with the C_∞ axis, and T_xy denotes the distance from this axis to the position of a fluorescent group. The translational and the rotational transformations between nearest protein neighbors are T_z and θ, respectively. The fluorophore undergoes local reorienting motions about an effective symmetry axis Z_D, which is transformed to the polymer fixed frame by Ω_DC = (α_DC, β_DC).

FIGURE 2

Polymerization and fluorescence properties of BODIPY-labeled β/γ-actin. (A) The increase in fluorescence upon polymerization of 8 μM β/γ-actin containing the fractions of fluorophore-labeled protein as indicated was recorded at 538 nm using an excitation wavelength of 485 nm (BODIPY fluorescence). (B) Pyrene fluorescence (excitation 365 nm, emission 410 nm) upon polymerization of 4 μM β/γ-actin containing 2% pyrene-actin and BODIPY-actin as indicated. (Inset) Recording of BODIPY fluorescence from the sample containing 1% of the actin labeled with BODIPY (A). Note that the BODIPY labeling does not influence the polymerization kinetics, and that the increase in relative fluorescence after polymerization reaches significantly higher values compared to pyrene-actin (see A and B). Panel C demonstrates the homogeneity of the labeled actin as seen by SDS-PAGE of 7.2 μg of labeled and 6.8 μg of nonlabeled protein, and panel D illustrates the visualization of BODIPY-labeled F-actin by fluorescence microscopy; (left) BODIPY-fluorescence (FITC filter) and (right) rhodamine-fluorescence due to double-staining with TRITC-phalloidin. β/γ-Actin at a concentration of 12 μM and consisting of 49% BODIPY-labeled actin was polymerized at room temperature and pipetted onto poly-L-lysine-coated coverslips followed by addition of 0.1 μM TRITC-phalloidin.

FIGURE 3

Fluorescence depolarization data obtained from the BODIPY-labeled F-actin. (Top panel) The results from the sample containing 0.1% BODIPY-labeled protein, showing the difference curve, D(t). The experimental response function is shown below the D(t) curve. The quality of a best-fit of the Maier-Saupe potential to D(t) is illustrated by the weighted residuals, wr(t). (Lower panel) The time-dependent fluorescence anisotropy, r(t), calculated from the depolarization data obtained for F-actin containing different mole fractions of BODIPY-labeled actin at 277 K. The dashed curve was simulated using the parameters obtained in the global analyses and assuming 100% labeling of the actin. The r(t)-decays curves in shading were obtained for G-actin at 277 (upper) and 293 K (lower).

FIGURE 4

The convergent behavior of the GA for a global fitting of all data on F-actin obtained at 277 K, with 486,000 BD trajectories and a population size of 30 individuals. The statistical test of the best fit (χ²) is plotted against the number of generations. Three independent fittings of the data are illustrated for three different initial seeds subjected to the random number generator.

FIGURE 5

Results from the global GA analysis of the fluorescence depolarization data on F-actin at 277 K, using 486,000 trajectories in the BD simulations. Three different F-actin samples containing 12.25, 24.5, and 49 mol % of BODIPY-labeled F-actin were analyzed. T_xy, T_z, α_DC, and β_DC denote the structural parameters defined in Fig. 1. (A) The pairs of α_DC and β_DC corresponding to the global minima. (B–D) The projections of χ²-values in the (α_DC, β_DC)-, (α_DC, θ)-, and (T_xy, T_z)-planes for the range 1 ≤ χ² ≤ 1.32.