An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: rewiring GFP.

The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore. Previously, it has been shown that the introduction of a second mutation (H148D) into S65T GFP allows the recovery of green emission, implying that ESPT is again possible. A similar recovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 is the proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emission following excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been explored through the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. The data are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicate very rapid (< 1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecond time scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionic mutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra that would correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrier hydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain of D148, with different potential energy surfaces for the ground and excited states. This model is consistent with recent high-resolution structural data in which the distance between the donor and acceptor oxygen atoms is < or = 2.4 A. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can be replaced by a single residue, an observation which, when fully explored, will add to our understanding of the various requirements for proton-transfer reactions within proteins.

Ultrafast Electronic and Vibrational Dynamics of Stabilized A State Mutants of the Green Fluorescent Protein (GFP): Snipping the Proton Wire.

Two blue absorbing and emitting mutants (S65G/T203V/E222Q and S65T at pH 5.5) of the green fluorescent protein (GFP) have been investigated through ultrafast time resolved infra-red (TRIR) and fluorescence spectroscopy. In these mutants, in which the excited state proton transfer reaction observed in wild type GFP has been blocked, the photophysics are dominated by the neutral A state. It was found that the A* excited state lifetime is short, indicating that it is relatively less stabilised in the protein matrix than the anionic form. However, the lifetime of the A* state can be increased through modifications to the protein structure. The TRIR spectra show that a large shifts in protein vibrational modes on excitation of the A* state occurs in both these GFP mutants. This is ascribed to a change in H-bonding interactions between the protein matrix and the excited state.

Ultrafast structural dynamics in BLUF domains: transient infrared spectroscopy of AppA and its mutants.

The structural dynamics following photoexcitation of a photosensing BLUF (blue light sensing using FAD) domain protein have been investigated by ultrafast transient infrared spectroscopy. Specifically, the transcriptional antirepressor AppA from Rhodobacter sphaeroides has been studied in the light and dark adapted forms and in photoactive and inactive mutants W104F and Q63L. A transient absorption has been observed at 1666 cm(-1) which is a marker mode for the photoactive state of the protein. This instantaneously formed transient is tentatively assigned to a vibrational mode of a protein residue modified through its interaction with the excited state of the chromophore. A plausible candidate consistent with the mutant studies is the carbonyl stretch of the Q63 amide side chain. These results suggest that modification of the strength of protein chromophore H-bonded interactions is the primary step in the BLUF domain photocycle. No new species were observed to be formed during the first nanosecond. Measurement of the ultrafast ground state recovery showed that the excited state of light adapted AppA is strongly quenched compared to the dark adapted state. It is proposed that the reorganization which occurs to form the signaling state is favorable to electron-transfer quenching.

Proton relay reaction in green fluorescent protein (GFP): Polarization-resolved ultrafast vibrational spectroscopy of isotopically edited GFP.

The complex transient vibrational spectra of wild type (wt) GFP have been assigned through polarization anisotropy measurements on isotopically edited proteins. Protein chromophore interactions modify considerably the vibrational structure, compared to the model chromophore in solution. An excited-state vibrational mode yields information on excited-state electronic structure. The proton relay pathway is characterized in more detail, and the protonation of the remote E222 residue is shown to occur in a concerted step. Modifications to protein vibrational modes are shown to occur following electronic excitation, and the potential for these to act as a trigger to the proton relay reaction is discussed.

Ultrafast vibrational spectroscopy of the flavin chromophore.

Ultrafast time-resolved infrared (TRIR) spectra of flavin adenine dinucleotide (FAD) and the anion of lumiflavin (Lf-) are described. Ground-state recovery and excited-state decay of FAD reveal a common dominant ultrafast relaxation and a minor slower component. The Lf- transient lacks a fast component. No intermediate species are observed, suggesting that the quenching mechanism is internal conversion promoted by interaction of the adenine and isoalloxazine rings in FAD. Modes are assigned, and the potential for extension of the TRIR method to photoactive proteins is discussed.

Pure surface plasmon resonance enhancement of the first hyperpolarizability of gold core-silver shell nanoparticles.

We report the optical second harmonic (SH) response from gold core-silver shell nanoparticles supported at a liquid-liquid interface in the spectral region where the second harmonic generation (SHG) frequency is resonant with the surface plasmon (SP) resonance excitation of the nanoparticles. We compare these results with that obtained by classical linear optical absorption spectroscopy and show that the nonlinear optical response is dominated by the SP resonance enhancement with negligible contributions from the interband transitions. As a result, the SH spectrum exhibits two clear SP resonance bands attributed to the two SP resonances of the composite nanostructure formed by the gold core-silver shell nanoparticles. Absolute values of the hyperpolarizabilities are measured by hyper Rayleigh scattering (HRS) and compared that of pure gold nanoparticles. The hyperpolarizability measured at a harmonic energy of 3.0 eV, enhanced through excitation of the high energy SP resonance of the nanoparticle, increases with the silver content whereas the hyperpolarizability measured at a harmonic energy of 2.4 eV, enhanced through the excitation of the low energy SP resonance of the nanoparticle, decreases because of the shift of this resonance away from the harmonic frequency. The hyperpolarizability determined by HRS and the square root of the SHG intensities, scaling with the nanoparticle hyperpolarizability, have similar trends with respect to the silver content indicative of closely related adsorption properties yielding similar surface concentrations at the liquid-liquid interface.

Wavelength dependence of the hyper Rayleigh scattering response from gold nanoparticles.

The wavelength dependence of the quadratic hyperpolarizability of 11 nm diam gold nanoparticles, is reported as measured by hyper Rayleigh scattering. An important photoluminescence background underlying the hyper Rayleigh signal is observed, a contribution attributed to radiative electron-hole recombinations following multiphoton excitation favored by adsorbed organic compound like citrate on the surface of the nanoparticles. The absolute value of the quadratic hyperpolarizability of gold spherical nanoparticles is determined and a strong enhancement is observed for harmonic frequencies in resonance with the dipolar surface plasmon excitation. No contribution of the interband transition is observed. The absolute values reported, beta(C)=5.1x10(-26) esu at the second harmonic energy 2.39 eV, have been measured with femtosecond long laser pulse, and are 1 order of magnitude weaker that the one previously reported with nanosecond long pulses. This difference can be related to similar measurements performed on the second order hyperpolarizability of gold nanoparticles and may be attributed to different electronic relaxation regimes. Finally, the spectrum of the quadratic hyperpolarizability is compared to the theoretically expected one.

Hyper-Rayleigh scattering of gold nanorods and their relationship with linear assemblies of gold nanospheres.

The surface plasmon enhanced hyper-Rayleigh scattering light collected from an aqueous solution of gold nanorods is reported. A non negligible part of the signal is attributed to a photoluminescence background attributed to the electron hole recombination following multiphoton excitation of d-valence band electrons into the sp-conduction band. This radiative relaxation process is likely favored by the presence of the organic species adsorbed at the surface of the nanorods. The absolute value for the hyperpolarisability of nanorods is also compared by the external reference method to that of para-nitroaniline and found to be rather large although an absolute value cannot be given because the exact number density of the gold nanorods is unaccessible. This value is however compared with values reported for linear assemblies of gold spherical nanoparticles and further support the simple model of gold metal ellipsoids to describe the hyper-Rayleigh light intensities. The polarisation analysis of the hyper-Rayleigh scattering light is also determined for gold nanorods and compared to the expected one for gold nanospheres. For the latter spheres, the weakness of the signal intensities precludes a definite comparison with the model. On the opposite, for the nanorods, the polarisation dependence of the hyper-Rayleigh scattered light clearly deviates from the one expected for nanospheres.