Ultrafast excited-state isomerization in phytochrome revealed by femtosecond stimulated Raman spectroscopy

Photochemical interconversion between the red-absorbing (P_r) and the far-red-absorbing (P_fr) forms of the photosensory protein phytochrome initiates signal transduction in bacteria and higher plants. The P_r-to-P_fr transition commences with a rapid Z-to-E photoisomerization at the C₁₅C₁₆ methine bridge of the bilin prosthetic group. Here, we use femtosecond stimulated Raman spectroscopy to probe the structural changes of the phycocyanobilin chromophore within phytochrome Cph1 on the ultrafast time scale. The enhanced intensity of the C₁₅–H hydrogen out-of-plane (HOOP) mode, together with the appearance of red-shifted CC stretch and NH in-plane rocking modes within 500 fs, reveal that initial distortion of the C₁₅C₁₆ bond occurs in the electronically excited I* intermediate. From I*, 85% of the excited population relaxes back to P_r in 3 ps, whereas the rest goes on to the Lumi-R photoproduct consistent with the 15% photochemical quantum yield. The C₁₅–H HOOP and skeletal modes evolve to a Lumi-R-like pattern after 3 ps, thereby indicating that the C₁₅C₁₆ Z-to-E isomerization occurs on the excited-state surface.     

Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode

The ultrabright femtosecond X-ray pulses provided by X-ray free-electron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this “probe-before-destroy” approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. Kβ_1,3 XES spectra of Mn^II and Mn₂^III,IV complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to >100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shot-by-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.     

Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy

Raman spectroscopy is a newly developed, noninvasive preclinical imaging technique that offers picomolar sensitivity and multiplexing capabilities to the field of molecular imaging. In this study, we demonstrate the ability of Raman spectroscopy to separate the spectral fingerprints of up to 10 different types of surface enhanced Raman scattering (SERS) nanoparticles in a living mouse after s.c. injection. Based on these spectral results, we simultaneously injected the five most intense and spectrally unique SERS nanoparticles i.v. to image their natural accumulation in the liver. All five types of SERS nanoparticles were successfully identified and spectrally separated using our optimized noninvasive Raman imaging system. In addition, we were able to linearly correlate Raman signal with SERS concentration after injecting four spectrally unique SERS nanoparticles either s.c. (R² = 0.998) or i.v. (R² = 0.992). These results show great potential for multiplexed imaging in living subjects in cases in which several targeted SERS probes could offer better detection of multiple biomarkers associated with a specific disease.     

Measurement of c-axis angular orientation in calcite (CaCO3) nanocrystals using X-ray absorption spectroscopy

We demonstrate that the ability to manipulate the polarization of synchrotron radiation can be exploited to enhance the capabilities of X-ray absorption near-edge structure (XANES) spectroscopy, to include linear dichroism effects. By acquiring spectra at the same photon energies but different polarizations, and using a photoelectron emission spectromicroscope (PEEM), one can quantitatively determine the angular orientation of micro- and nanocrystals with a spatial resolution down to 10 nm. XANES-PEEM instruments are already present at most synchrotrons, hence these methods are readily available. The methods are demonstrated here on geologic calcite (CaCO(3)) and used to investigate the prismatic layer of a mollusk shell, Pinctada fucata. These XANES-PEEM data reveal multiply oriented nanocrystals within calcite prisms, previously thought to be monocrystalline. The subdivision into multiply oriented nanocrystals, spread by more than 50°, may explain the excellent mechanical properties of the prismatic layer, known for decades but never explained.     

Analysis of CCR5-Delta 32 and CCR2-V64I polymorphisms in a cohort of Spanish HCV patients using real-time polymerase chain reaction and fluorescence resonance energy transfer technologies

Summary.  Nowadays it is clear that chemokine–chemokine receptor interactions are important in chronic hepatitis C virus (HCV) infection. The objective of the present study was to elucidate the involvement of the CCR5 -Δ32 and CCR2- V64I polymorphisms in the response to the HCV infection, as well as in the histological damage and the outcome of the infection. A cohort of 139 patients with hepatitis C and 100 healthy blood donors were analysed for both polymorphisms using real-time polymerase chain reaction (PCR) and LightCycler^TM technology. We have detected the CCR5 -Δ32 allele in 15 of 278 HCV chromosomes (5.4%) and 15 of 200 control chromosomes (7.5%). The CCR2- V64I allele was present in 24 of 278 HCV chromosomes (8.6%) and 19 of 200 control chromosomes (9.5%). Analysis of the histological parameters showed no statistical significance when comparing the patients carrying the variants vs the cases with the wild-type allele. Our results seem to indicate that the CCR5 -Δ32 and CCR2 -V64I polymorphisms are not related to the response to HCV infection, histological damage and outcome of infection in our cohort of Spanish HCV patients.