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In response to stress small organic compounds termed osmolytes are ubiquitously accumulated in all cell types to regulate the intracellular solvent quality and to counteract the deleterious effect on the stability and function of cellular proteins. Given the evidence that destabilization of the native state of a protein either by mutation or by environmental changes triggers the aggregation in the neurodegenerative pathologies, the modulation of the intracellular solute composition with osmolytes is an attractive strategy to stabilize an aggregating protein. Here we report the effect of three natural osmolytes on the in vivo and in vitro aggregation landscape of huntingtin exon 1 implicated in the Huntington's disease. Trimethylamine N-oxide (TMAO) and proline redirect amyloid fibrillogenesis of the pathological huntingtin exon 1 to nonamyloidogenic amorphous assemblies via two dissimilar molecular mechanisms. TMAO causes a rapid formation of bulky amorphous aggregates with minimally exposed surface area, whereas proline solubilizes the monomer and suppresses the accumulation of early transient aggregates. Conversely, glycine betaine enhances fibrillization in a fashion reminiscent of the genesis of functional amyloids. Strikingly, none of the natural osmolytes can completely abrogate the aggregate formation; however, they redirect the amyloidogenesis into alternative, nontoxic aggregate species. Our study reveals new insights into the complex interactions of osmoprotectants with polyQaggregates.
We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are beta-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.
Efficient triplet exciton emission has allowed improved operation of organic light-emitting diodes (LEDs). To enhance the device performance, it is necessary to understand what governs the motion of triplet excitons through the organic semiconductor. Here, we have investigated triplet diffusion using a model compound that has weak energetic disorder. The Dexter-type triplet energy transfer is found to be thermally activated down to a transition temperature T- T, below which the transfer rate is only weakly temperature dependent. We show that above the transition temperature, Dexter energy transfer can be described within the framework of Marcus theory. We suggest that below T-T, the nature of the transfer changes from phonon-assisted hopping to quantum-mechanical tunneling. The lower electron-phonon coupling and higher electronic coupling in the polymer compared to the monomer results in an enhanced triplet diffusion rate.
Microelectrodes modified with electropolymerized plumbagin (PLG) were used for the generation of superoxide radical (O-2(center dot-)) and hydrogen peroxide (H2O2) during oxygen reduction reaction (ORR) in an aqueous medium, specifically in serum-free cell culture media. This is enabled by the specific design of a polymer film on the microelectrode. The generation and diffusion of O-2(center dot-) during electrocatalytic ORR at a positionable PLG polymer-modified microelectrode was followed by fluorescence microscopy with the selective dye 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and by amperometric detection using a cytochrome c-modified electrode at + 0.13 V. H2O2 production, either by direct oxygen reduction or as product of O-2(center dot-) disproportionation, was monitored by the reaction with Amplex UltraRed. The PLG polymer-modified microelectrodes were used to expose mammalian B6-RPE07 retinal cells to defined local fluxes of reactive oxygen species (ROS), and cellular responses and morphological alterations were observed. The use of a controllable source of ROS opens many possibilities to study how living cells respond to the presence of a certain flux of specific ROS.
Vibrationally resolved fluorescence spectra of four angular [N]phenylenes were recorded with laser excited Shpol’skii spectroscopy (LESS) in an n-octane matrix at 10 K. In general, the same vibrational frequencies were observed in the fluorescence excitation and emission spectra, indicating that the geometries of ground and electronically excited state are very similar. Because of intensity borrowing from the S2 state, vibrations of two different symmetries were observed in the fluorescence excitation spectra of angular [3]phenylene and zig-zag[5]phenylene. This finding allowed the location of the S2 state for these compounds. DFT calculations(RB3LYP/6-31G*) of the ground state vibrational frequencies were made. The calculated vibrational modes were in reasonably good agreement with the experimental data. A new very low-frequency vibration of approximately 100 cm-1 was predicted and experimentally confirmed for all [N]phenylenes investigated. This vibration seems to be unique for [N]phenylenes and is attributed to an in-plane movement of the carbon backbone.
Deuteration effects on the vibronic structure of the emission and excitation spectra of triangular [4]phenylene (D3h[4]phenylene) were studied using laser-excited Shpolskii spectroscopy (LESS) in an octane matrix at 4.2 K. For correct assignment of the vibrational modes, the experimental results were compared with calculated frequencies (B3LYP/6-31G*). CH vibrations were identified by their characteristic isotopic shifts in the spectra of deuterated triangular [4]phenylenes. Two CC stretching modes, at 100 cm–1 and 1176 cm–1, suitable as probes for bond strength changes in the excited state, were identified. The isotope effect on the internal conversion rates of triangular [4]phenylene was evaluated from measurements of temperature dependent lifetime. Isotope dependency and the magnitude of the internal conversion rates indicate that internal conversion in triangular [4]phenylene is most likely induced by CH vibrations. The results obtained by LESS and lifetime measurements were compared with PM3 PECI calculations of the excited state structure. The theoretical results and the relation between ground and excited state vibration energies of the 1176 cm–1 probe vibration indicate a reduction of bond alternation of the central cyclohexatriene ring in the excited state.
Deuteration effects on the vibronic structure of the emission and excitation spectra of triangular [ 4] phenylene (D-3h [4]phenylene) were studied using laser-excited Shpol'skii spectroscopy (LESS) in an octane matrix at 4.2 K. For correct assignment of the vibrational modes, the experimental results were compared with calculated frequencies (B3LYP/6-31G*). CH vibrations were identified by their characteristic isotopic shifts in the spectra of deuterated triangular [4]phenylenes. Two CC stretching modes, at 100 cm(-1) and 1176 cm(-1), suitable as probes for bond strength changes in the excited state, were identified. The isotope effect on the internal conversion rates of triangular [4] phenylene was evaluated from measurements of temperature dependent lifetime. Isotope dependency and the magnitude of the internal conversion rates indicate that internal conversion in triangular [4] phenylene is most likely induced by CH vibrations. The results obtained by LESS and lifetime measurements were compared with PM3 PECI calculations of the excited state structure. The theoretical results and the relation between ground and excited state vibration energies of the 1176 cm(-1) probe vibration indicate a reduction of bond alternation of the central cyclohexatriene ring in the excited state