Session W26: Focus Session: Biological Photophysics

Direct Observation of Thymine Dimer Repair in DNA by Photolyase

Departments of Physics, Chemistry, and Biochemistry, Programs of Biophysics, Chemical Physics, and Biochemistry, The Ohio State University, Columbus, 191 West Woodruff Avenue, OH 43210. Photolyase uses light energy to split ultraviolet-induced cyclobutane pyrimidine dimers in damaged DNA, but its molecular mechanism has never been directly revealed. We report here the direct mapping of catalytic processes through femtosecond synchronization of the enzymatic dynamics with the repair function. We observed direct electron transfer from the excited flavin cofactor to the dimer in 170 ps and back electron transfer from the repaired thymines in 560 ps. Both reactions are strongly modulated by active-site solvation to achieve maximum repair efficiency. These results show that the photocycle of DNA repair by photolyase is through a radical mechanism and completed on subnanosecond time scale at the dynamic active site with no net electron change in redox states of the flavin cofactor.   

Near-infrared femtosecond laser assisted cell membrane permeabilization

The controlled delivery of membrane impermeable molecules into single living cells (micro-injection) is important for a variety of applications such genomics, proteomics or drug screening and testing. Recently, it has been demonstrated that opto-injection with tightly focused ($\sim $300nm) femtosecond (fs) laser pulses at near-infrared (nir) wavelengths (700-1100nm) has the potential to create highly localized transient pores in single living cells with high cell survival rates and transfection efficiency. We have investigated the creation of transient pores in single living BAEC cells by focused fs nir laser pulses dependent on the incident laser intensity by dye uptake studies. Our experimental data agree very well with the experimentally and theoretically determined thresholds for laser-induced plasma formation and LIB. We observe that pore creation is observed for laser intensities of 4.0x10$^{12}$W/cm$^{2}$ and higher. For laser intensities above 3.3x10$^{13}$W/cm$^{2}$ BAEC cells are irreversibly destroyed. Within these two limits the pore size increases logarithmically with increasing laser intensity. This functional dependence is explained by considering the Gaussian intensity distribution across the laser focal spot. The physical understanding of the relationship between pore size and laser intensity allows the control of the number of molecules delivered into a cell per unit time through the control of the pore size.   

Biological Photophysics with Small Photons: Terahertz measurements of the protein dynamical Transition

Protein tertiary structural vibrations lay in the terahertz frequency range. These motions are associated with the large scale conformational motions necessary for function. Previously we have shown protein terahertz dielectric response is sensitive to protein conformation, hydration, oxidation state, ligand binding and folding. In this talk I will focus on our measurements of the 200 K dynamical transition and how these results fit into the slaved solvent model. Further I will discuss how these measurements along with hydration dependence measurements demonstrate the water beyond the first solvation shell continues to deviate from bulk water behavior. This work was supported by ACS grant PRF 39554-AC6, NSF CAREER grant PHY-0349256 and NSF IGERT grant DGE0114330.   

DFT calculation of photo-induced charge transfer in organic molecule

We propose a method for obtaining the charge transfer time for a chromophore-donor-acceptor system from density functional theory. Our calculations are done on a porphyrin-carotene-C$_{60}$ molecular triad. The geometry of the large molecular triad was optimized at the all-electron level using GGA. We are considering single electron excitations, the energies of which are obtained using a newly developed approach. The electronic dipolar transition probabilities are calculated from Einstein's A and B coefficients. However, in real systems the polarization effects play an important role in the transfer process since the charge separated states can possess huge dipole moments. The stabilization of the large dipole state can be calculated from the classical dipole-dipole interaction and the polarizability of the surrounding medium. The stabilization of the dipole states is an important aspect which dominates the charge transfer process and therefore the rise time. The efficiency of the molecule as a solar energy converter will also be discussed.   

Structural colours in blue-banded bee

Periodic, micro-textured biological materials are ubiquitous in nature. Electromagnetic waves at different frequencies are selectively reflected by such materials. This phenomenon is the origin of structural colours observed in variety of insects. In this work, we analyze the mechanisms that lead to the bluish-green colour of the blue-banded bee feathers. The reflection spectrum of the blue-banded bee feather was calculated by the transfer matrix method (TMM). The reflection peaks found are compatible within the experimental data. In addition to Bragg scattering, guided resonance has been observed in our theoretical calculation, which leads to a novel understanding of the structural colours in blue-banded bees.   

Wavelength-Dependent Conformational Changes of Collagen in Mid-IR Ablation

Single pulses of the Mark-III free electron laser have been used to ablate porcine corneas at a fluence of 240 J/cm$^{2}$ and wavelengths of 2.77 and 6.45 $\mu $m. As previously characterized, the non-volatile ablation debris shows evidence of wavelength-dependent collagen fragmentation. We have measured micro-Raman spectra of the debris and the ablation crater to determine if any wavelength-dependent conformational changes have taken place. Comparison of the spectra from two different wavelengths shows that a 938 cm$^{-1}$ Raman band -- assignable to the peptide C$_{C=O}$-C$_{\alpha }$ stretch of collagen -- loses substantial intensity during 6.45-$\mu $m ablation, but not in 2.77-$\mu $m ablation. This intensity decrease may be associated with a reduction of collagen triple-helix structure. Other spectral techniques yield mixed results; signatures for the loss of triple-helix structure are evident in UV-CD and some aspects of $^{13}$C-NMR spectra, but not in FTIR spectra. Raman measurements on thermally-treated corneal slices display similar changes at high temperatures, suggesting that higher protein temperatures are reached during ablation at 6.45 $\mu $m when compared to 2.77 $\mu $m. These observations suggest that any pre-vaporization loss of protein structural integrity includes not only collagen fragmentation, but also a loss of collagen triple-helix structure.   

Novel protection mechanisms against singlet oxygen formation by the Chl $a$ molecule in the cytochrome $b_{6}f$ complex of oxygenic photosynthesis

A Chl molecule is known to produce highly toxic singlet oxygen under light illumination as a result of energy transfer from its triplet excited state to oxygen. To prevent that, a carotenoid is typically positioned close to a Chl molecule ($\sim $4 $\AA$) in Chl containing proteins to ensure rapid triplet-triplet energy transfer from Chl to carotenoid. Surprisingly, the X-ray structures of the cytochrome $b_{6}f$ complex show that the $\beta$ -carotene is much too far from the only Chl $a$ found in this complex to provide effective protection by the usual triplet-triplet energy transfer mechanism. Our optical femtosecond time resolved experiments on diluted samples as well as on the single crystals of the $b_{6}f$ complex suggest that the Chl $a$ is protected by two novel mechanisms: (i) the yield of the Chl $a$ triplet state formation is reduced through electron-transfer exchange with the nearby amino acid residues, and (ii) a long distance triplet energy transfer to carotenoid mediated by a third mobile molecule (NSF MCB- 0516939, NIH GM-38323).   

Electrostatic Steering of Functional Dynamics in GFP

Distinctive photodynamic properties of the green fluorescent protein (GFP) from the jellyfish \emph{A. victoria} result from charge transfer processes involving the autocatalytically generated chromophore. We investigate structural changes in response to chromophore photoionization at cryogenic temperatures, using both X-ray crystallography and polarized infrared measurements on oriented single crystals. These measurements identify conformational changes of Gln 69, Cys 70, and an associated H-bonded cluster of internal water molecules in the chromophore environment. These structural changes take place at 100 K, far below the ``dynamical transition'' traditionally regarded as enabling functional protein motions. This contrasts with the prevailing view that the rigid interior of the GFP $\beta$-barrel sterically inhibits nonradiative processes. Instead, we propose that rapid rearrangements of the chromophore environment enhance the fluorescence quantum yield by stabilizing the abruptly altered charge distribution in the radiative state. We suggest that the conformational response to charge transfer influences two fundamental and useful spectroscopic properties of GFP---the large frequency separation between excitation and emission and the efficient fluorescence.   

Terahertz Dielectric Response of Photoactive Yellow Protein (PYP): Influence of Conformational-Vibrational State during Photocycle and Hydration Effects

Protein conformational change alters flexibility and conformational-vibrational modes that occur on a picosecond or sub-picosecond time scale. Terahertz dielectric measurements are sensitive to protein flexibility as they directly probe the density of states of these vibrational modes. Using terahertz time-domain spectroscopy, we measured the dielectric response of PYP thin films as a function of resting and photointermediate state. The absorbance increases smoothly as a function of frequency while the index of refraction exhibits no frequency dependence. A sharp transition in the dielectric response of the ground state is observed at 86{\%} relative humidity (r.h.), corresponding to the point where the protein film has lost $\sim $50 water molecules relative to a 100{\%} r.h. environment. Similar transitions observed for hen egg white lysozyme and cytochrome c correspond to the filling point of the first hydration shell.   

Novel Photo-Protecticon Mechanisms in Chlorosomes from Green Sulfur Bacterium \textit{Chlorobium Tepidum}

Chlorosome is the largest known photosynthetic light-harvesting antenna complex that incorporates thousands of bacteriochlorophtylls (BChl) and carotenoids (Car) in a closely packed quasi-regular structure. BChl is known to produce highly toxic singlet oxygen as the result of energy transfer from their excited triplet states to oxygen molecules. It has been proposed that that the carotenoids in chlorosome serve both light harvesting and photo-protection functions, transferring light excitations to nearby BChl and simultaneously quenching excited triplet state of BChl. However, experiments indicate that photoprotective role of carotenoids in chlorosomes is not as extensive as expected---photo-degradation of carotenoid-free mutants occurs only twice faster than photo-degradation of native complexes. An additional non-conventional photoprotection mechanism must exist in chlorosomes. The possible nature of this mechanism is discussed based on optical kinetic measurements of BChl and Car excited states.   

Hydration Dependence of Energy Relaxation Time for Cytochrome C

Hydration plays a critical role in protein dynamics. Here we consider the effects of hydration on energy relaxation for an electronically excited heme protein cytochrome c. We measure the hydration dependence of energy relaxation time of cytochrome C films after photoexcitation in the Soret regionusing two-color pump/probe time resolved transmission measurements. Thin films were prepared from cytochrome C/ Trizma buffer solutions and mounted in a hydration controlled cell. We used 400nm ($\sim $3 mW) to pump the B band and 800 nm ($\sim $1 mW) to probe the III band. The III band corresponds to the charge-transfer transition between heme $\pi $ and iron d orbital, and is assigned to the ground electronic state of the heme. Therefore this band can be used to probe the ground state population. Three separate dynamic components were observed: a very fast transient $\tau _{1} \quad \sim $ 200 fs; a several hundred femtosecond component ($\tau _{2})$; and a recovery of the ground state absorption($\tau _{3})$. We find $\tau _{3}$ apparently decreases with decreasing hydration while $\tau _{1}$ and $\tau _{2}$ are independent of hydration.