Delmar S. Larsen

Uncovering the hidden ground state of green fluorescent protein

The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFPs hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I1 and I2, has been unveiled. I1 and I2 absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I2 intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen/deuterium exchange of the protein leads to a significant increase of the I1 and I2 lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology.

Photoisomerization and Photoionization of the Photoactive Yellow Protein Chromophore in Solution

Dispersed pump-dump-probe spectroscopy has the ability to characterize and identify the underlying ultrafast dynamical processes in complicated chemical and biological systems. This technique builds on traditional pump-probe techniques by exploring both ground- and excited-state dynamics and characterizing the connectivity between constituent transient states. We have used the dispersed pump-dump-probe technique to investigate the ground-state dynamics and competing excited-state processes in the excitation-induced ultrafast dynamics of thiomethyl p-coumaric acid, a model chromophore for the photoreceptor photoactive yellow protein. Our results demonstrate the parallel formation of two relaxation pathways (with multiple transient states) that jointly lead to two different types of photochemistry: cis-trans isomerization and detachment of a hydrated electron. The relative transition rates and quantum yields of both pathways have been determined. We find that the relaxation of the photoexcited chromophores involves multiple, transient ground-state intermediates and the chromophore in solution does not generate persistent photoisomerized products, but instead undergoes photoionization resulting in the generation of detached electrons and radicals. These results are of great value in interpreting the more complex dynamical changes in the optical properties of the photoactive yellow protein.

Influence of intramolecular vibrations in third-order, time-domain resonant spectroscopies. II. Numerical calculations

We model recent experimental wavelength dependent Three Pulse Photon Echo Peak Shift (WD-3PEPS) and Transient Grating (WD-TG) signals considering both solvation dynamics and vibrational contributions. We present numerical simulations of WD-3PEPS and WD-TG signals of two probe molecules: Nile Blue and N,N-bisdimethylphenyl-2,4,6,8-perylenetetracarbonyl diamide to investigate the influence of intramolecular vibrations in the signals. By varying the excitation wavelength, we show that the different initial conditions for the vibrational wave packets significantly affect the signals, especially through the contributions associated with high frequency modes, often neglected in experimental analyses. We show that the temporal properties of both WD-TG and WD-3PEPS signals display sensitivities to both the excitation wavelength and the vibronic structure of the specific probe molecule used. Several mechanisms for generating vibronic modulations in the signals are discussed and their effects on the signals are des...

Three pulse photon echo studies of nondipolar solvation: Comparison with a viscoelastic model

Three pulse stimulated photon echo peak shift (3PEPS) measurements were used to probe the solvation of a quadrupolar solute in three room temperature nondipolar solvents; benzene, CCl4, and CS2, and the results were compared with those for two polar solvents, methanol and acetonitrile, and one weakly polar solvent, toluene. Our data reveal three distinct solvent dynamical time scales; a sub-100 fs ultrafast component attributed to inertial motions, a slow (∼2–3 ps) component attributed to structural relaxation, and an intermediate time scale (∼600 fs) of uncertain origin. The six solvents were chosen to reflect a range of possible interactions, but exhibit similar dynamics, suggesting that similar mechanisms may be at work or that different mechanisms may exist, but occur on similar time scales. A viscoelastic continuum solvation model proposed to describe nonpolar solvation [J. Phys. Chem. A 102, 17 (1998)] was used for a preliminary analysis of our data.

Influence of intramolecular vibrations in third-order, time-domain resonant spectroscopies. I. Experiments

This is the first in a two-paper series that investigates the influence of intramolecular vibrational modes on nonlinear, time-domain, electronically resonant signals. Both Transient Grating (TG) and Three Pulse Photon Echo Peak Shift (3PEPS) signals were collected from several probe molecules: Nile Blue, N,N-bis-dimethylphenyl-2,4,6,8-perylenetetracarbonyl diamide, and Rhodamine 6G dissolved in different solvents: benzene, dimethylsulfoxide, and acetonitrile. The effects of excitation of different vibronic transitions on the electronically resonant signals were identified by comparing signals collected with laser pulses at different excitation wavelengths. In the 3PEPS profiles, we find that excitation on the blue edge of the absorption spectrum causes a decreased initial peak shift values and more rapid initial decays, whilst in the TG signals, the magnitude of the “coherent spike” is strongly wavelength dependent. Additional thermally activated vibronic effects were studied via temperature dependent 3P...

Femtosecond ligand/core dynamics of microwave-assisted synthesized silicon quantum dots in aqueous solution.

A microwave-assisted reaction has been developed to produce hydrogen-terminated silicon quantum dots (QDs). The Si QDs were passivated for water solubility via two different methods: hydrosilylation produced 3-aminopropenyl-terminated Si QDs, and a modified Stöber process produced silica-encapsulated Si QDs. Both methods produce water-soluble QDs with maximum emission at 414 nm, and after purification, the QDs exhibit intrinsic fluorescence quantum yield efficiencies of 15 and 23%, respectively. Even though the QDs have different surfaces, they exhibit nearly identical absorption and fluorescence spectra. Femtosecond transient absorption spectroscopy was used for temporal resolution of the photoexcited carrier dynamics between the QDs and ligand. The transient dynamics of the 3-aminopropenyl-terminated Si QDs is interpreted as a formation and decay of a charge-transfer (CT) excited state between the delocalized π electrons of the carbon linker and the Si core excitons. This CT state is stable for ~4 ns before reverting back to a more stable, long-living species. The silica-encapsulated Si QDs show a simpler spectrum without CT dynamics.

Femtosecond Photodynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. 1. Forward Dynamics

Phytochromes are well-known red/far-red photosensory proteins that utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to detect the ratio of red to far-red light. Cyanobacteriochromes (CBCRs) are related photosensory proteins with a bilin-binding GAF domain, but much more diverse spectral sensitivity, with five recognized subfamilies of CBCRs described to date. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing ground state, like the familiar P(r) state of phytochromes, and a green-absorbing photoproduct (P(g)). Here, we examine the ultrafast forward photodynamics of the red/green CBCR NpR6012g4 from the NpR6012 locus of the nitrogen-fixing cyanobacterium Nostoc punctiforme. Using transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics that induces the forward reaction (red-absorbing to green-absorbing), which we interpret as arising from ground-state heterogeneity. Excited-state decays with lifetimes of 55 and 345 ps generate the primary photoproduct (Lumi-R), and the fastest decay (5 ps) did not produce Lumi-R. Although the photoinduced kinetics of Npr6012g4 is comparable with that of the Cph1 phytochrome isolated from Synechocystis cyanobacteria, NpR6012g4 exhibits a ≥2-3-fold higher photochemical quantum yield. Understanding the structural basis of this enhanced quantum yield may prove to be useful in increasing the photochemical efficiency of other bilin-based photosensors.

Intrinsic optical heterodyne detection of a two-dimensional fifth order Raman response

Intrinsic optical heterodyne detection is applied to femtosecond two-dimensional (2-D) vibrational spectroscopy of the Raman active inter- and intramolecular modes of liquid CS 2 between 1 and 1000 cm -t. Heterodyne detection is accomplished with a novel wave vector matching geometry that allows detection of the cross term between the third and fifth order response, R(3)R (5). The increase in the bandwidth and sensitivity through this detection technique will allow the measurement of 2-D temporal or spectral responses for a wide variety of systems in this frequency range.

Packing Dependent Electronic Coupling in Single Poly(3-hexylthiophene) H- and J-Aggregate Nanofibers

Nanofibers (NFs) of the prototype conjugated polymer, poly(3-hexylthiophene) (P3HT), displaying H- and J-aggregate character are studied using temperature- and pressure-dependent photoluminescence (PL) spectroscopy. Single J-aggregate NF spectra show a decrease of the 0-0/0-1 vibronic intensity ratio from ~2.0 at 300 K to ~1.3 at 4 K. Temperature-dependent PL line shape parameters (i.e., 0-0 energies and 0-0/0-1 intensity ratios) undergo an abrupt change in the range of ~110-130 K suggesting a change in NF chain packing. Pressure-dependent PL lifetimes also show increased contributions from an instrument-limited decay component which is attributed to greater torsional disorder of the P3HT backbone upon decreasing NF volume. It is proposed that the P3HT alkyl side groups change their packing arrangement from a type I to type II configuration causing a decrease in J-aggregate character (lower intrachain order) in both temperature- and pressure-dependent PL spectra. Chain packing dependent exciton and polaron relaxation and recombination dynamics in NF aggregates are next studied using transient absorption spectroscopy (TAS). TAS data reveal faster polaron recombination dynamics in H-type P3HT NFs indicative of interchain delocalization whereas J-type NFs exhibit delayed recombination suggesting that polarons (in addition to excitons) are more delocalized along individual chains. Both time-resolved and steady-state spectra confirm that excitons and polarons in J-type NFs are predominantly intrachain in nature that can acquire interchain character with small structural (chain packing) perturbations.

Initial photo-induced dynamics of the photoactive yellow protein chromophore in solution

Abstract The initial photoinduced dynamics of thiomethyl p -coumaric acid (TMpCA) in solution has been studied with dispersed time-resolved pump–probe spectroscopy with a ∼100 fs instrument response and extending over a wavelength range of ∼300 to ∼600 nm. TMpCA is a model chromophore for the intrinsic chromophore found in photoactive yellow protein (PYP). Stimulated emission from the chromophore is quenched on a timescale similar to chromophore within the PYP protein. A product state absorption is also observed and is formed earlier than the relaxation of the excited state and that of an observed transient intermediate.