Time-resolved fluorescence anisotropy and molecular dynamics analysis of a novel GFP homo-FRET dimer

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 coexpressed 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 dimerization, in which 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 a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, 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 (〈E FRET exp〉 = 0.25 ± 0.05) and those calculated from the molecular dynamics simulations (〈E FRET MD〉 = 0.18 ± 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors 〈κ 2〉 = 0.17 ± 0.16 and 〈|κ|〉 = 0.35 ± 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time- and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET. </p

Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging (FLIM) on the nanosecond time scale, and highlight some applications of these techniques

Noise-Corrected Principal Component Analysis of fluorescence lifetime imaging data

International audienceFluorescence Lifetime Imaging (FLIM) is an attractivemicroscopy method in the life sciences, yielding informa-tion on the sample otherwise unavailable through inten-sity-based techniques. A novel Noise-Corrected PrincipalComponent Analysis (NC-PCA) method for time-domainFLIM data is presented here. The presence and distribu-tion of distinct microenvironments are identified at lowerphoton counts than previously reported, without requir-ing prior knowledge of their number or of the dye’s decaykinetics. A noise correction based on the Poisson statisticsinherent to Time-Correlated Single Photon Counting isincorporated. The approach is validated using simulateddata, and further applied to experimental FLIM data ofHeLa cells stained with membrane dye di-4-AN-EPPDHQ. Two distinct lipid phases were resolved in thecell membranes, and the modification of the order para-meters of the plasma membrane during cholesterol deple-tion was also detected

PRODAN differentially influences its local environment

PRODAN influences its local environment at the nanoscale differently between ordered and disordered phases as shown by MD simulations.</p

Genetically encoded sensors of protein hydrodynamics and molecular proximity

The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1′-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin