Christian Schumann

Ultrafast Infrared Spectroscopy of Riboflavin: Dynamics, Electronic Structure, and Vibrational Mode Analysis

Femtosecond time-resolved infrared spectroscopy was used to study the vibrational response of riboflavin in DMSO to photoexcitation at 387 nm. Vibrational cooling in the excited electronic state is observed and characterized by a time constant of 4.0 +/- 0.1 ps. Its characteristic pattern of negative and positive IR difference signals allows the identification and determination of excited-state vibrational frequencies of riboflavin in the spectral region between 1100 and 1740 cm (-1). Density functional theory (B3LYP), Hartree-Fock (HF) and configuration interaction singles (CIS) methods were employed to calculate the vibrational spectra of the electronic ground state and the first singlet excited pipi* state as well as respective electronic energies, structural parameters, electronic dipole moments and intrinsic force constants. The harmonic frequencies of the S 1 excited state calculated by the CIS method are in satisfactory agreement with the observed band positions. There is a clear correspondence between computed ground- and excited-state vibrations. Major changes upon photoexcitation include the loss of the double bond between the C4a and N5 atoms, reflected in a downshift of related vibrations in the spectral region from 1450 to 1720 cm (-1). Furthermore, the vibrational analysis reveals intra- and intermolecular hydrogen bonding of the riboflavin chromophore.

Subpicosecond Midinfrared Spectroscopy of the Pfr Reaction of Phytochrome Agp1 from Agrobacterium tumefaciens

Phytochromes are light-sensing pigments found in plants and bacteria. For the first time, the P(fr) photoreaction of a phytochrome has been subject to ultrafast infrared vibrational spectroscopy. Three time constants of 0.3 ps, 1.3 ps, and 4.0 ps were derived from the kinetics of structurally specific marker bands of the biliverdin chromophore of Agp1-BV from Agrobacterium tumefaciens after excitation at 765 nm. VIS-pump-VIS-probe experiments yield time constants of 0.44 ps and 3.3 ps for the underlying electronic-state dynamics. A reaction scheme is proposed including two kinetic steps on the S(1) excited-state surface and the cooling of a vibrationally hot P(fr) ground state. It is concluded that the upper limit of the E-Z isomerization of the C(15) = C(16) methine bridge is given by the intermediate time constant of 1.3 ps. The reaction scheme is reminiscent of that of the corresponding P(r) reaction of Agp1-BV as published earlier.

Primary photoinduced protein response in bacteriorhodopsin and sensory rhodopsin II.

Essential for the biological function of the light-driven proton pump, bacteriorhodopsin (BR), and the light sensor, sensory rhodopsin II (SRII), is the coupling of the activated retinal chromophore to the hosting protein moiety. In order to explore the dynamics of this process we have performed ultrafast transient mid-infrared spectroscopy on isotopically labeled BR and SRII samples. These include SRII in D(2)O buffer, BR in H(2)(18)O medium, SRII with (15)N-labeled protein, and BR with (13)C(14)(13)C(15)-labeled retinal chromophore. Via observed shifts of infrared difference bands after photoexcitation and their kinetics we provide evidence for nonchromophore bands in the amide I and the amide II region of BR and SRII. A band around 1550 cm(-1) is very likely due to an amide II vibration. In the amide I region, contributions of modes involving exchangeable protons and modes not involving exchangeable protons can be discerned. Observed bands in the amide I region of BR are not due to bending vibrations of protein-bound water molecules. The observed protein bands appear in the amide I region within the system response of ca. 0.3 ps and in the amide II region within 3 ps, and decay partially in both regions on a slower time scale of 9-18 ps. Similar observations have been presented earlier for BR5.12, containing a nonisomerizable chromophore (R. Gross et al. J. Phys. Chem. B 2009, 113, 7851-7860). Thus, the results suggest a common mechanism for ultrafast protein response in the artificial and the native system besides isomerization, which could be induced by initial chromophore polarization.

The Differential Action of Cytocalasin D in T-tubular Remodelling of Ventricular Myocytes

Cytocalasin D (cytoD) is a fungal metabolite that inhibits cytokinesis by blocking formation of contractile microfilament structures resulting in multi-nucleated cells, reversible inhibition of cell movement, and the induction of cellular extrusion. However, in muscle cells reports are controversial and vary from actin stabilization to actin disruption at concentrations of 40 µM. The aim of this study was to investigate the putative cytoD effects on adult cardiomyocytes in culture and how it can be used as a pharmacological tool in single cell models. This investigation is based on our optimized serum-free culture method for adult rat ventricular myocytes.Initially, for global calcium transients we performed repetitive dose response relationships over the time course of 6 days to identify the optimal cytoD concentration. At this identified concentration we analyzed action potentials, calcium handling (incl. post rest behavior and caffein induced calcium release) and cellular contractility. In addition, we investigated cytoD-mediated changes in the T-tubular system and actin cytoskeleton by confocal and STED microscopy, respectively.In contrast to previous reports we were able to identify a cytoD concentration that resulted in a high degree of morphological and functional conservation of properties found in freshly isolated cells over a time course of one week.In conclusion our findings represent a further significant improvement for single cell models of cardiomyocytes, enabling chronic stimulation and an extended time period for adenoviral protein expression. The identified relationship between actin filament modulation and prevention of T-tubular loss might shed new light on the loss of T-tubules in cardiac diseases.This work was supported by the DFG (KFO196) and the Medical Faculty (HOMFOR).

Stimulated emission depletion microscopy for imaging of engineered and biological nanostructures

The investigation of interactions between engineered nanostructures and biological systems is a key component in the assessment of potential environmental and health implications due to the increasing application of nanotechnology. Combining the high specificity of bioconjugate fluorescence labeling techniques with the sub-diffraction resolution of Stimulated Emission Depletion (STED) microscopy and state-of-the-art nonlinear image restoration allows the imaging of these interactions on the length scales demanded by the interaction partners. In this article, we give an overview of the experimental approach and discuss its implications on the biological interpretation of the resulting fluorescence micrographs.