Session J34: Focus Session: Impact of Ultrafast Lasers III: Biophysics

Probing Electronic Excitations in Molecules by Coherent Multidimensional UV and X Ray Spectroscopy

Two-dimensional ultraviolet (2DUV) spectra of protein backbone (far UV) and side chains (near UV) provide new insights into the protein structures, dynamics and functions. Simulated chirality-induced 2DUV spectra reveal characteristic patterns of protein secondary structures and allow monitoring the aggregation mechanism of amyloid fibrils and predicting the aggregation propensity of peptides. Time-domain experiments that employ sequences of attosecond x-ray pulses in order to probe electronic and nuclear dynamics in molecules are made possible by newly developed bright coherent ultrafast sources of soft and hard x-rays. By creating multiple core holes at selected atoms and controlled times it should be possible to study the dynamics and correlations of valence electrons as they respond to these perturbations. The stimulated x-ray Raman spectrum of \textit{trans}-N-methylacetamide and Cysteine at the Nitrogen, Sulfur and the Oxygen K-edges in response to two soft x-ray pulses is calculated by treating the core excitations at the Hartree--Fock static-exchange level (STEX) level. The signal is interpreted in terms of the dynamics of valence electronic wave packets prepared and detected in the vicinity of (either the nitrogen or the oxygen) atom. The evolving electronic charge density and as electronic coherences are visualized using a basis set of time-dependent natural orbitals. Effects of orbital relaxation upon core excitations are resolved. A two-dimensional extension of the technique that involves a sequence of three resonant Raman pulses will be presented. Extensions to multidimensional spectroscopy with photoelectron detection are proposed.   

Conformation of self-assembled porphyrin dimers in liposome vesicles by phase-modulation 2D fluorescence spectroscopy

By applying a phase-modulation fluorescence approach to 2D electronic spectroscopy (PM-2D FS), we studied the conformation-dependent exciton coupling of a porphyrin dimer embedded in a phospholipid bilayer membrane. Our measurements specify the relative angle and separation between interacting electronic transition dipole moments and thus provide a detailed characterization of dimer conformation. PM-2D FS produces 2D spectra with distinct optical features, similar to those obtained using 2D photon-echo spectroscopy. Specifically, we studied magnesium meso tetraphenylporphyrin dimers, which form in the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes. Comparison between experimental and simulated spectra show that although a wide range of dimer conformations can be inferred by either the linear absorption spectrum or the 2D spectrum alone, consideration of both types of spectra constrain the possible structures to a ``T-shaped'' geometry. These experiments establish the PM-2D FS method as an effective approach to elucidate chromophore dimer conformation.   

2-Dimensional Fluorescence Spectroscopy: Determining the Temperature-Dependent Conformations of Porphyrin Dimers and Nucleic Acids

I will describe spectroscopic studies on a covalently-linked zinc tetraphenylporphyrin dimer embedded in a phospholipid bilayer membrane. Using phase-modulation 2-Dimensional Fluorescence Spectroscopy (2D FS, a fluorescence-detected version of 2D electronic spectroscopy) along with linear absorption and fluorescence spectroscopy, it was found that the dimer adopts two predominant conformations in the membrane, and that the relative populations of these two states change as a function of temperature. Simultaneously fitting the linear absorption spectrum and the 2D FS spectra at four different excitation wavelengths revealed a wealth of information about these two states, including their relative populations, relative fluorescence quantum yields, the strength of the exciton coupling present in each state, and the approximate angles between the electronic transition dipole moments of the two porphyrins. Ongoing analysis focuses on elucidating the relaxation and energy transfer dynamics of this system through the population time dependence of the 2D spectra. Finally, I will present preliminary results from experiments in which 2D FS was performed with ultraviolet excitation to study the conformations of DNA constructs labeled with a fluorescent analogue of guanine.   

Exploring Relaxation Processes in Components of DNA with UV Nonlinear Spectroscopy

Underlying photoinduced relaxation in DNA is a complex world of solute-solvent interactions and fluctuations in the geometries of macromolecules. Electronic excitations are rapidly deactivated by nuclear motions through conical intersections, thereby suppressing the formation of lesions (e.g., thymine dimers) known to inhibit cellular function. At the instant following internal conversion, the bases are left in ``hot'' quantum states, wherein a subset of vibrational modes possess a highly non-equilibrium distribution of excitation quanta. The transfer of this energy to the surrounding also involves intriguing fundamental physics. We examine these processes in small components of DNA by conducting femtosecond laser spectroscopies at cryogenic temperatures. Our experiments utilize several recent advances in nonlinear optics. Parametric processes in argon gas are used to generate 25fs pulse durations at 265nm. These short pulses are employed in a variety of measurements (e.g., transient grating, 2D photon echo, fluorescence down-conversion) with the goal of understanding relaxation mechanisms. Our data suggest that excited state deactivation in DNA is quite sensitive to the exchange of vibrational energy between the bases and segments of the backbone.   

Early optical response of fluorescent molecules studied by synthetic optically delayed pulses

The early optical response of fluorescent molecules in solution is probed by pairs of collinear pulse replicas. Two approaches are followed. First approach mimics an interferometer, replicating interference as a function of time delay between the pulses. For the second approach, each pulse spans the entire laser bandwidth, sharing no common frequencies with the second pulse, thus no interference is observed between the pulses. In both cases, the pair of pulses is delayed with attosecond resolution to study IR 144. Both fluorescence at right angles or the stimulated emission along the output beam as a function of time delay is monitored. At high intensities when approximately 10{\%} of the dye molecules are excited, the second pulse can stimulate emission from molecules excited by the first pulse, thereby giving rise to interference fringes every 2.66 fs. When the pulse replicas are generated by multiple independent comb shaping, it is evident that the interference fringes for stimulated emission bear an out of phase relationship with those observed from fluorescence and have a maxima at time zero. This is masked with conventional pulse replicas that interfere.   

Quantum mechanical light harvesting mechanisms in photosynthesis

More than 10 million billion photons of light strike a leaf each second. Incredibly, almost every red-coloured photon is captured by chlorophyll pigments and initiates steps to plant growth. Last year we reported that marine algae use quantum mechanics in order to optimize photosynthesis [1], a process essential to its survival. These and other insights from the natural world promise to revolutionize our ability to harness the power of the sun. In a recent review [2] we described the principles learned from studies of various natural antenna complexes and suggested how to utilize that knowledge to shape future technologies. We forecast the need to develop ways to direct and regulate excitation energy flow using molecular organizations that facilitate feedback and control--not easy given that the energy is only stored for a billionth of a second. In this presentation I will describe new results that explain the observation and meaning of quantum-coherent energy transfer. \\[4pt] [1] Elisabetta Collini, Cathy Y. Wong, Krystyna E. Wilk, Paul M. G. Curmi, Paul Brumer, and Gregory D. Scholes, ``Coherently wired light-harvesting in photosynthetic marine algae at ambient temperature'' Nature 463, 644-648 (2010).\\[0pt] [2] Gregory D. Scholes, Graham R. Fleming, Alexandra Olaya-Castro and Rienk van Grondelle, ``Lessons from nature about solar light harvesting'' Nature Chem. 3, 763-774 (2011).   

Temperature dependence of proton transfer kinetics in the green fluorescent protein

In green fluorescent protein (GFP), near UV photoexcitation leads to proton transfer from the chromophore phenolic oxygen along a proton ``wire'' consisting of an internal water molecule, Ser205 and Glu222. Using transient absorption kinetics, the complete cycle, including the picosecond excited-state proton transfer, the nanosecond radiative emission, and the slower ground state proton back-transfer reactions have been studied holistically as a function of temperature. This experiment was performed for both the hydrogenated and deuterated forms of GFP. We have extracted the Arrhenius prefactors and activation energy barriers for both the forward and back proton transfer kinetics. A large kinetic isotope effect for the ground state proton back-transfer has been observed at high temperatures suggesting that tunneling plays an important role. At lower temperatures the data suggest a cross-over to a different pathway for the back-transfer reaction. To investigate this hypothesis we studied the E222D mutant of GFP, which substitutes aspartate for glutamate on the proton wire. The H/D kinetics of this mutant explicitly test for the source of proton donors and indicate that proton transfer proceeds along the same pathway in the native protein at room temperature.