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Time-resolved fluorescence anisotropy and molecular dynamics analysis of a novel GFP homo-FRET dimer
Biophysical Journal ( IF 3.4 ) Pub Date : 2021-01-01 , DOI: 10.1016/j.bpj.2020.11.2275 Yurema Teijeiro-Gonzalez 1 , Alessandro Crnjar 1 , Andrew J Beavil 2 , Rebecca L Beavil 2 , Jakub Nedbal 1 , Alix Le Marois 3 , Carla Molteni 1 , Klaus Suhling 1
Biophysical Journal ( IF 3.4 ) Pub Date : 2021-01-01 , DOI: 10.1016/j.bpj.2020.11.2275 Yurema Teijeiro-Gonzalez 1 , Alessandro Crnjar 1 , Andrew J Beavil 2 , Rebecca L Beavil 2 , Jakub Nedbal 1 , Alix Le Marois 3 , Carla Molteni 1 , Klaus Suhling 1
Affiliation
Förster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are co-expressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerisation, where its signature consists of a very short component at the beginning of the anisotropy decay. In this work we present an approach to study GFP homo-FRET via combination of time-resolved fluorescence anisotropy, the stretched exponential decay model and molecular dynamics (MD) simulations. We characterise a new FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model = 0.25 ± 0.05) and those calculated from the MD simulations (= 0.18 ± 0.14). The relative dipole orientation between the GFPs is best described by the orientational-factors = 0.17 ± 0.16 and |k| = 0.35 ± 0.20, contextualised within a static framework, where the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time and polarisation-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET.
更新日期：2021-01-01
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