The effect of local dynamics of Atto 390‐labeled lysozyme on fluorescence anisotropy modeling

Abstract

Fluorescence anisotropy decay is a popular optical technique to study the structure, size, shape and even functions of biomolecules. The method measures the time dependence of the depolarization of a fluorophore and is therefore sensitive to the changes in the rotational motion (e.g., aggregation and binding) or changes in the mobility of segments of biopolymers (such as the ones associated with tertiary structure changes). Fluorescence anisotropy decay often requires the use of fluorescent dyes that need to be covalently attached to the biomolecule. The location of the attachment on the biomolecule (e.g., a protein) and the linker used, affect the mobility of the dye and its anisotropy decay. With this study we have combined the experimental data with molecular dynamic simulations to offer a more correct interpretation of the fluorescence anisotropy decay of a popular fluorescent dye (Atto 390) attached to the N-terminus of Hen Egg White Lysozyme (HEWL). Our model shows how the use of relatively simple molecular dynamics computation to simulate the motion of the dye, provide a model to interpret the experimental fluorescence anisotropy decay that yields a better estimate of the hydrodynamic radius of HEWL. The improvement is due to a more detailed description of the segmental motion of the dye attached to the protein.

On the Interpretation of subtilisin Carlsberg Time-Resolved Fluorescence Anisotropy Decays: Modeling with Classical Simulations

“…The Wobbling-in-a-Cone Model and the Interpretation of Janot et al The reorientational dynamics of a fluorescent probe with partially restricted motions can be modeled by the wobbling-in-a-cone model. 20,42,46 This model establishes that the fluorophore can diffuse freely inside a cone with semiangle θ max while attached to a larger molecular group with slower reorientational motions. In this case, the anisotropy can be expressed as 20…”

“…The reorientational dynamics of a fluorescent probe with partially restricted motions can be modeled by the wobbling-in-a-cone model. ,, This model establishes that the fluorophore can diffuse freely inside a cone with semiangle θ max while attached to a larger molecular group with slower reorientational motions. In this case, the anisotropy can be expressed as where ϕ Trp is the correlation time for the motion of the Trp (the fluorophore) in the protein and ϕ p is the correlation time of the global motion of the protein.…”

Integrated NMR, Fluorescence, and Molecular Dynamics Benchmark Study of Protein Mechanics and Hydrodynamics

“…For instance, fluorescence spectroscopy and MD simulations have been jointly applied to various problems, which is also based on the fact that the analysis of fluorescence anisotropy decay is a tool to elucidate internal motion and diffusion of biomolecules and is a topic of broad theoretical consideration. − MD can be used to validate the fluorescence anisotropy for studying the flexibility of proteins, , but these approaches suffered from lack of adequate fluorescence analysis, suboptimal dye selection, and relying on not validated dye force fields. More often, when MD is combined with fluorescence, it is used either to explain the molecular basis of a fluorescence observable ,− or to validate the parametrization of dye models , for further modeling of FRET-based structures. − Further information and examples for the combination of fluorescence spectroscopy and MD can be found in a review by Stella et al, which highlights the complementarity of both techniques and the resulting potential for their synergistic application. The combination of NMR and fluorescence spectroscopy was applied to small organic molecules to estimate precise absolute diffusion coefficients , where the fluorescence was used to estimate the effects of protein concentration.…”