Enhancement of the Fluorescence of the Blue Fluorescent Proteins by High Pressure or Low Temperature

Abstract

Green fluorescent proteins bearing the Y66H mutation exhibit strongly blue-shifted fluorescence excitation and emission spectra. However, these blue fluorescent proteins (BFPs) have lower quantum yields of fluorescence (Phi(f) approximately 0.20), which is believed to stem from the increased conformational freedom of the smaller chromophore. We demonstrate that suppression of chromophore mobility by increasing hydrostatic pressure or by decreasing temperature can enhance the fluorescence quantum yield of these proteins without significantly affecting their absorption properties or the shape of the fluorescence spectra. Analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes. Temperature-dependent fluorescence measurements reveal two barriers ( approximately 19 and 3 kJ/mol, respectively) for the transition into nonfluorescing states. These steps are probably linked with dissociation of the hydrogen bond between the chromophore and His148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state, respectively. The chromophore's hydrogen-bond equilibrium at room temperature is dominated by entropic effects, while below approximately 200 K the balance is enthalpy-driven.

Dynamic Stokes shift in green fluorescent protein variants

“…Both the synthetic GFP chromophore (20) and the chromophore in denatured protein (21) exhibit a very low fluorescence quantum yield, likely because the chromophore undergoes isomerization and nonfluorescent relaxation when the solvent is flexible. Consistent with this theory, the isolated chromophore fluorescence quantum yield increases dramatically if the solvent is frozen (22), and low temperature and/or high pressure can increase the quantum yield for GFP variants with intrinsically low quantum yields, such as blue fluorescent protein (23). Thus, a high fluorescence quantum yield, which is desirable for native function (and biotechnology), and the conditions of protein flexibility needed to observe a dynamic Stokes shift appear to be mutually exclusive for GFP variants.…”

Photophysics of the blue fluorescent protein

“…For comparison, the neutral form of GFP chromophore has a minimal distance of 5600 cm À1 between S0 and S1 states [11]. The energy gap law [12] predicts a fast nonradiative transition to the ground state (shortest BFP fluorescence decay component has a lifetime of 1.2 ps at 300 K [2]), followed by efficient relaxation on the steep potential energy surface. Analogous dependence of the potential energy on double bond twist has been found for the chromophore of wild type GFP [9].…”

“…BFP has been used as a donor molecule for fluorescence resonant energy transfer (FRET) [1], but the low quantum yield (20%) has prevented its widespread usage. Recent experiments have shown [2] that decrease in temperature or application of high hydrostatic pressure enhance fluorescence quantum yield remarkably. The highly nonexponential fluorescence decay [2][3][4] with lifetime span from 1.2 ps to 2 ns indicates that the chromophore of BFP has several conformations and/or different pathways of quenching of excited state.…”

“…Recent experiments have shown [2] that decrease in temperature or application of high hydrostatic pressure enhance fluorescence quantum yield remarkably. The highly nonexponential fluorescence decay [2][3][4] with lifetime span from 1.2 ps to 2 ns indicates that the chromophore of BFP has several conformations and/or different pathways of quenching of excited state. In the present contribution, we attempt to explain the above-mentioned nonexponential decay by semi-empirical calculations of the excited state surfaces.…”

Efficient Photoconversion Distorts the Fluorescence Lifetime of GFP in Confocal Microscopy: A Model Kinetic Study on Mutant Thr203Val

“…[10] Softening of the interior of the protein barrel was detected by the temperature dependence of t fl in blue fluorescent protein. [11] Conformational heterogeneity in cyan fluorescent protein (CFP) was found to be the origin of t fl heterogeneity, which subsequently was overcome in cerulean by point mutations. [12,13] t fl can also be manipulated by isosteric replacement of amino acids.…”