Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins

Abstract

Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G͞S72A, are discovered in the 10 ؊6 -10 ؊3 -s time range, by using fluorescence correlation spectroscopy at 10 ؊8 M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over Ϸ6 ؋ 10 2 to 5 ؋ 10 6 W͞cm 2 . These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of Ϸ10 ؊3 . Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10-to 100-s time range. The independence of these two bright-dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

“…We compare our interpretation of the results further in the Supporting Information, with reference to fluorescence correlation spectroscopy (FCS) of Schwille et al (4), lowtemperature absorption spectroscopy of Creemers et al (3) and single-molecule optical switching by Beltram and colleagues (7,8). More recently Blum et al (40) have shown further heterogeneity in YFP fluorescence and have detected species that exhibit blue-and red-shifted emission compared with the dominant 527 nm emission.…”

“…They have also attracted the attention of spectroscopists owing to their complex photochemistry (2)(3)(4)(5). In particular, those in the YFP class (i.e.…”

Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

“…Correlation analysis, on the other hand, provides quantitative information about the timescales of conformational fluctuations; however, its interpretation is often complicated by its strong model dependence, and by the presence of optical artifacts. [28] Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single-molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states.…”

Photostability of green and yellow fluorescent proteins with fluorinated chromophores, investigated by fluorescence correlation spectroscopy

“…Even at moderate intensities, the contrast C is constant and independent of further intensity increase. Such behaviour indicates light-driven cis-trans isomerization [29,30]. This is also true for the fluorinated proteins in this study; however, the absolute values of C and accordingly [B] differ (eq.2).…”