Evidence for a transfer-to-trap mechanism of fluorophore concentration quenching in lipid bilayers.

Autor:	Meredith SA; School of Physics and Astronomy, University of Leeds, Leeds, UK; Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK., Kusunoki Y; Graduate School of Agricultural Science and Biosignal Research Center, Kobe University, Kobe, Japan., Evans SD; School of Physics and Astronomy, University of Leeds, Leeds, UK., Morigaki K; Graduate School of Agricultural Science and Biosignal Research Center, Kobe University, Kobe, Japan., Connell SD; School of Physics and Astronomy, University of Leeds, Leeds, UK; Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK., Adams PG; School of Physics and Astronomy, University of Leeds, Leeds, UK; Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK. Electronic address: p.g.adams@leeds.ac.uk.
Jazyk:	angličtina
Zdroj:	Biophysical journal [Biophys J] 2024 Sep 17; Vol. 123 (18), pp. 3242-3256. Date of Electronic Publication: 2024 Jul 22.
DOI:	10.1016/j.bpj.2024.07.026
Abstrakt:	It is important to understand the behaviors of fluorescent molecules because, firstly, they are often utilized as probes in biophysical experiments and, secondly, they are crucial cofactors in biological processes such as photosynthesis. A phenomenon called "fluorescence quenching" occurs when fluorophores are present at high concentrations, but the mechanisms for quenching are debated. Here, we used a technique called "in-membrane electrophoresis" to generate concentration gradients of fluorophores within a supported lipid bilayer, across which quenching was expected to occur. Fluorescence lifetime imaging microscopy (FLIM) provides images where the fluorescence intensity in each pixel is correlated to fluorescence lifetime: the intensity provides information about the location and concentration of fluorophores and the lifetime reveals the occurrence of energy-dissipative processes. FLIM was used to compare the quenching behavior of three commonly used fluorophores: Texas Red (TR), nitrobenzoaxadiazole (NBD), and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). FLIM images provided evidence of quenching in regions where the fluorophores accumulated, but the degree of quenching varied between the different fluorophores. The relationship between quenching and concentration was quantified and the "critical radius for trap formation," representing the relative quenching strength, was calculated as 2.70, 2.02, and 1.14 nm, for BODIPY, TR, and NBD, respectively. The experimental data support the theory that quenching takes place via a "transfer-to-trap" mechanism which proposes, firstly, that excitation energy is transferred between fluorophores and may reach a "trap site," resulting in immediate energy dissipation, and, secondly, that trap sites are formed in a concentration-dependent manner. Some previous work suggested that quenching occurs only when fluorophores aggregate, or form long-lived dimers, but our data and this theory argue that traps may be "statistical pairs" of fluorophores that exist only transiently. Our findings should inspire future work to assess whether these traps can be charge-transfer states, excited-state dimers, or something else. Competing Interests: Declaration of interests The authors declare that they have no competing interests. (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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