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.

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.

Ultrafast dynamics in complex fluids observed through the ultrafast optically-heterodyne-detected optical-Kerr-effect (OHD-OKE).

The ultrafast molecular dynamics of complex fluids have been recorded using the optically-heterodyne-detected optical-Kerr-effect (OHD-OKE). The OHD-OKE method is reviewed and some recent refinements to the method are described. Applications to a range of complex fluids, including microemulsions, polymer melts and solutions, liquid crystal and ionic liquids are surveyed.

Evidence from Raman spectroscopy that InhA, the mycobacterial enoyl reductase, modulates the conformation of the NADH cofactor to promote catalysis.

InhA, the enoyl reductase from Mycobacterium tuberculosis, catalyzes the NADH-dependent reduction of trans-2-enoyl-ACPs. In the present work, Raman spectroscopy has been used to identify catalytically relevant changes in the conformation of the nicotinamide ring that occur when NADH binds to InhA. For 4(S)-NADD, there is an 11 cm-1 decrease in the wavenumber of the C4-D stretching band (nuC-D) and a 50% decrease in the width of this band upon binding to InhA.

Temperature- and solvation-dependent dynamics of liquid sulfur dioxide studied through the ultrafast optical Kerr effect.

The ultrafast dynamics of liquid sulphur dioxide have been studied over a wide temperature range and in solution. The optically heterodyne-detected and spatially masked optical Kerr effect (OKE) has been used to record the anisotropic and isotropic third-order responses, respectively. Analysis of the anisotropic response reveals two components, an ultrafast nonexponential relaxation and a slower exponential relaxation.

Ring current effects in the active site of medium-chain Acyl-CoA dehydrogenase revealed by NMR spectroscopy.

Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the flavin-dependent oxidation of fatty acyl-CoAs to the corresponding trans-2-enoyl-CoAs. The interaction of hexadienoyl-CoA (HD-CoA), a product analogue, with recombinant pig MCAD (pMCAD) has been studied using (13)C NMR and (1)H-(13)C HSQC spectroscopy. Upon binding to oxidized pMCAD, the chemical shifts of the C1, C2, and C3 HD carbons are shifted upfield by 12.

Observation of excited-state proton transfer in green fluorescent protein using ultrafast vibrational spectroscopy.

The photodynamics of wtGFP have been studied by ultrafast time-resolved infrared spectroscopy (TIR). In addition to the expected bleaching and transient infrared absorption of bands associated with the chromophore, we observe the dynamics of the proton relay reaction in the protein. Protonation of a protein carboxylate group occurs on the tens of picoseconds time scale following photoexcitation.

Ultrafast dynamics in the dispersed phase of oil-in-water microemulsions: monosubstituted benzenes incorporated into dodecyltrimethylammonium bromide (DTAB) aqueous micelles.

Time-resolved optical Kerr effect spectroscopy has been used to probe the molecular environment afforded by the hydrophobic core of oil-in-water microemulsions. This was achieved by measuring the ultrafast dynamics of a series of benzene derivatives (benzonitrile, nitrobenzene, fluorobenzene, styrene, and toluene) incorporated as the oil phase within oil-in-water microemulsions and comparing them to the dynamics in neat liquid and the liquid diluted in nonpolar solvent. Polar and strongly interacting liquids (benzonitrile and nitrobenzene) showed dynamics in the microemulsion that are similar to those in the solution phase, while weakly interacting and mildly polar liquids (fluorobenzene, styrene and toluene) reveal dynamics more similar to those of the neat liquid.