Rotational motion of rhodamine 6G tethered to actin through oligo(ethylene glycol) linkers studied by frequency-domain fluorescence anisotropy

Abstract

Investigation of the rotational motion of a fluorescent probe tethered to a protein helps to elucidate the local properties of the solvent and protein near the conjugation site of the probe. In this study, we have developed an instrument for frequency-domain fluorescence (FDF) anisotropy measurements, and studied how the local properties around a protein, actin, can be elucidated from the rotational motion of a dye tethered to actin. Rhodamine 6G (R6G) was attached to Cys-374 using newly-synthesized R6G-maleimide with three different oligo(ethylene glycol) (OEG) linker lengths. The time-resolved anisotropy decay of R6G tethered to G-actin was revealed to be a combination of the two modes of the wobbling motion of R6G and the tumbling motion of G-actin. The rotational diffusion coefficient (RDC) of R6G wobbling was ~0.1 ns−1 at 20°C and increased with OEG linker length. The use of the three R6G-actin conjugates of different linker lengths was useful to not only figure out the linker length dependence of the rotational motion of R6G but also validate the analyses. In the presence of a cosolvent of glycerol, although the tumbling motion of G-actin was retarded in response to the bulk viscosity, the wobbling motion of R6G tethered to actin exhibited an increase of RDC as glycerol concentration increased. This finding suggests an intricate relationship between the fluid properties of the bulk solvent and the local environment around actin.

Cell-Sized Confinements Alter Molecular Diffusion in Concentrated Polymer Solutions Due to Length-Dependent Wetting of Polymers

“…Prior to the D R analysis inside dextran droplets, we confirmed that the effect of polarization of the excitation light is negligible in the diffusion measurements from the almost identical translational correlation time, τ D , in the bulk with or without the use of polarizers (Table S1 and Section S1). In addition, the values of the lifetime of the fluorescence molecule τ L and of τ R in the bulk without dextran are almost identical with the reference values . Accordingly, we obtained τ D inside the dextran droplets and plotted the normalized diffusion coefficients, D / D bulk , against R for systems with and without polarizers (Figure a).…”

“…In addition, the values of the lifetime of the fluorescence molecule τ L and of τ R in the bulk without dextran are almost identical with the reference values. 32 Accordingly, we obtained τ D inside the dextran droplets and plotted the normalized diffusion coefficients, D / D bulk , against R for systems with and without polarizers ( Figure 2 a). We again observe a slow translational diffusion of R6G inside small dextran droplets with R < 20 μm, as in the case of TAMRA ( Figure 1 e).…”