The peripheral light-harvesting antenna complex (LH2) of purple photosynthetic bacteria is an ideal testing ground for models of structure-function relationships due to its well-determined molecular structure and ultrafast energy deactivation. It has been the target for numerous studies in both theory and ultrafast spectroscopy; nevertheless, certain aspects of the convoluted relaxation network of LH2 lack a satisfactory explanation by conventional theories. For example, the initial carotenoid-to-bacteriochlorophyll energy transfer step necessary on visible light excitation was long considered to follow the Förster mechanism, even though transfer times as short as 40 femtoseconds (fs) have been observed. Such transfer times are hard to accommodate by Förster theory, as the moderate coupling strengths found in LH2 suggest much slower transfer within this framework. In this study, we investigate LH2 from Phaeospirillum (Ph.) molischianum in two types of transient absorption experiments-with narrowband pump and white-light probe resulting in 100 fs time resolution, and with degenerate broadband 10 fs pump and probe pulses. With regard to the split Qx band in this system, we show that vibronically mediated transfer explains both the ultrafast carotenoid-to-B850 transfer, and the almost complete lack of transfer to B800. These results are beyond Förster theory, which predicts an almost equal partition between the two channels.
- MeSH
- bakteriochlorofyly metabolismus MeSH
- časové faktory MeSH
- Fourierova analýza MeSH
- karotenoidy metabolismus MeSH
- lasery MeSH
- přenos energie * MeSH
- Proteobacteria metabolismus MeSH
- spektrofotometrie ultrafialová MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Lysine hydroxylation of type I collagen telopeptides varies from tissue to tissue, and these distinct hydroxylation patterns modulate collagen cross-linking to generate a unique extracellular matrix. Abnormalities in these patterns contribute to pathologies that include osteogenesis imperfecta (OI), fibrosis, and cancer. Telopeptide procollagen modifications are carried out by lysyl hydroxylase 2 (LH2); however, little is known regarding how this enzyme regulates hydroxylation patterns. We identified an ER complex of resident chaperones that includes HSP47, FKBP65, and BiP regulating the activity of LH2. Our findings show that FKBP65 and HSP47 modulate the activity of LH2 to either favor or repress its activity. BiP was also identified as a member of the complex, playing a role in enhancing the formation of the complex. This newly identified ER chaperone complex contributes to our understanding of how LH2 regulates lysyl hydroxylation of type I collagen C-telopeptides to affect the quality of connective tissues. © 2017 American Society for Bone and Mineral Research.
- MeSH
- biologické modely MeSH
- buněčné linie MeSH
- hmotnostní spektrometrie MeSH
- hydroxylace MeSH
- kolagen typu I metabolismus MeSH
- lidé MeSH
- lysin metabolismus MeSH
- lysinhydroxylasa metabolismus MeSH
- multiproteinové komplexy metabolismus MeSH
- mutace genetika MeSH
- myši MeSH
- peptidy metabolismus MeSH
- povrchová plasmonová rezonance MeSH
- prokolagen metabolismus MeSH
- proteiny tepelného šoku HSP47 metabolismus MeSH
- proteiny teplotního šoku metabolismus MeSH
- proteiny vázající takrolimus metabolismus MeSH
- stabilita enzymů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Energy relaxation in light-harvesting complexes has been extensively studied by various ultrafast spectroscopic techniques, the fastest processes being in the sub-100-fs range. At the same time, much slower dynamics have been observed in individual complexes by single-molecule fluorescence spectroscopy (SMS). In this work, we use a pump-probe-type SMS technique to observe the ultrafast energy relaxation in single light-harvesting complexes LH2 of purple bacteria. After excitation at 800 nm, the measured relaxation time distribution of multiple complexes has a peak at 95 fs and is asymmetric, with a tail at slower relaxation times. When tuning the excitation wavelength, the distribution changes in both its shape and position. The observed behavior agrees with what is to be expected from the LH2 excited states structure. As we show by a Redfield theory calculation of the relaxation times, the distribution shape corresponds to the expected effect of Gaussian disorder of the pigment transition energies. By repeatedly measuring few individual complexes for minutes, we find that complexes sample the relaxation time distribution on a timescale of seconds. Furthermore, by comparing the distribution from a single long-lived complex with the whole ensemble, we demonstrate that, regarding the relaxation times, the ensemble can be considered ergodic. Our findings thus agree with the commonly used notion of an ensemble of identical LH2 complexes experiencing slow random fluctuations.
- MeSH
- bakteriochlorofyly chemie účinky záření MeSH
- čas MeSH
- fluorescenční spektrometrie metody MeSH
- konfokální mikroskopie MeSH
- lasery MeSH
- neparametrická statistika MeSH
- normální rozdělení MeSH
- přenos energie * MeSH
- Rhodopseudomonas chemie MeSH
- světlo MeSH
- světlosběrné proteinové komplexy chemie účinky záření MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- srovnávací studie MeSH
Potentiometric and spectrophotometric pH-titrations of the lesinurad for three consecutive dissociation constants determination were compared. Lesinurad is a selective inhibitor of uric acid reabsorption as part of a combination of medicines to treat high levels of uric acid in blood, also called hyperuricemia. Nonlinear regression of the pH-spectra with REACTLAB and SQUAD84 and of the pH-titration curve with ESAB determined three multiple close dissociation constants. The protonation scheme of lesinurad was suggested. A sparingly soluble anion L- of lesinurad was protonated to the still soluble species LH, LH2+ and LH32+ in pure water. Three consecutive thermodynamic dissociation constants were estimated pKTa1 = 2.09, pKTa2 = 4.25, pKTa3 = 6.58 at 25 °C and pKTa1 = 1.96, pKTa2 = 4.16, pKTa3 = 6.32 at 37 °C by UV-metric spectra analysis. The graph of molar absorption coefficients shows that the spectrum of species LH2+ and LH vary in colour, while protonation of chromophore LH2+ to LH32+ has less influence on chromophores in the lesinurad molecule. Three multiple thermodynamic dissociation constants of 1 × 10-4 M lesinurad were determined by the pH-metric analysis pKTa1 = 2.39, pKTa2 = 3.47, pKTa3 = 6.17 at 25 °C and pKTa1 = 2.08, pKTa2 = 3.29, pKTa3 = 6.03 at 37 °C. The values of enthalpy ΔH0(pKa1) = 19.19 kJ mol-1, ΔH0(pKa2) = 13.29 kJ mol-1, ΔH0(pKa3) = 38.39 kJ mol-1, show the dissociation process is endothermic. The positive values of ΔG0(pKa1) = 11.93 kJ mol-1, ΔG0(pKa2) = 24.26 kJ mol-1, ΔG0(pKa3) = 37.56 kJ mol-1 at 25 °C indicate that the dissociation process of pKa2 is not spontaneous, which was confirmed by its value of entropy ΔS0(pKa1) = 24.37 J mol-1, ΔS0(pKa2) = -36.79 J mol-1, ΔS0(pKa3) = 2.79 J mol-1. Three macro-dissociation constants of lesinurad and protonation locations were predicted by MARVIN and ACD/Percepta.
- MeSH
- farmaceutická chemie MeSH
- koncentrace vodíkových iontů MeSH
- potenciometrie přístrojové vybavení metody MeSH
- spektrofotometrie ultrafialová přístrojové vybavení metody MeSH
- termodynamika * MeSH
- thioglykoláty chemie MeSH
- triazoly chemie MeSH
- urikosurika chemie MeSH
- Publikační typ
- časopisecké články MeSH
RC-LH1-PufX complexes from a genetically modified strain of Rhodobacter sphaeroides that accumulates carotenoids with very long conjugation were studied by ultrafast transient absorption spectroscopy. The complexes predominantly bind the carotenoid diketospirilloxanthin, constituting about 75% of the total carotenoids, which has 13 conjugated C=C bonds, and the conjugation is further extended to two terminal keto groups. Excitation of diketospirilloxanthin in the RC-LH1-PufX complex demonstrates fully functional energy transfer from diketospirilloxanthin to BChl a in the LH1 antenna. As for other purple bacterial LH complexes having carotenoids with long conjugation, the main energy transfer route is via the S2-Qx pathway. However, in contrast to LH2 complexes binding diketospirilloxanthin, in RC-LH1-PufX we observe an additional, minor energy transfer pathway associated with the S1 state of diketospirilloxanthin. By comparing the spectral properties of the S1 state of diketospirilloxanthin in solution, in LH2, and in RC-LH1-PufX, we propose that the carotenoid-binding site in RC-LH1-PufX activates the ICT state of diketospirilloxanthin, resulting in the opening of a minor S1/ICT-mediated energy transfer channel.
- MeSH
- bakteriochlorofyly metabolismus MeSH
- fluorescenční spektrometrie MeSH
- karotenoidy metabolismus MeSH
- kinetika MeSH
- počítačové zpracování signálu MeSH
- přenos energie * MeSH
- Rhodobacter sphaeroides metabolismus MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- xanthofyly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Baricitinib is a drug used for the treatment of rheumatoid arthritis. It is a selective and reversible inhibitor of Janus kinases 1 and 2, which play an important role in signalling the pro-inflammatory pathway activated in autoimmune disorders such as rheumatoid arthritis. The pH-spectrophotometric and pH-potentiometric titrations allowed the measurement of three or four successive dissociation constants of Baricitinib. Baricitinib neutral LH2 molecule was able to protonate into two soluble cations LH42+, LH3+ and dissociate into two soluble anions LH- and L2- in pure water. The graph of molar absorption coefficients of differently protonated species versus wavelength indicated that the spectra εL, εLH, εLH2 were the nearly the same for these species and that the spectra εLH4 and εLH3 were also similar. In the pH range from 2-13, four pKa´s of spectra analysis were reliably estimated by REACTLAB at I =0.0020 mol. dm-3 values pKTa1 = 3.07, pKTa2 = 3.87, pKTa3 = 6.27, pKTa4 = 12.78 at 25 °C and pKTa1 = 3.00, pKTa2 = 3.79, pKTa3 = 6.12, pKTa4 = 12.75 at 37 °C. Potentiometric pH-titration analysis for a higher concentration of 1 × 10-3 mol. dm-3 estimated with ESAB at I =0.0001 mol. dm-3 values pKTa1 = 3.69, pKTa2 = 3.81, pKTa3 = 4.73 at 25 °C and pKTa1 = 3.62, pKTa2 = 3.73, pKTa3 = 4.43 at 37 °C. Molar enthalpy ΔH°, molar entropy ΔS° and Gibbs free energy ΔG° were calculated from the spectra using a dependence ln K to 1/T.
- MeSH
- azetidiny * MeSH
- entropie MeSH
- koncentrace vodíkových iontů MeSH
- puriny MeSH
- pyrazoly MeSH
- sulfonamidy MeSH
- termodynamika MeSH
- Publikační typ
- časopisecké články MeSH
Carotenoids are known to offer protection against the potentially damaging combination of light and oxygen encountered by purple phototrophic bacteria, but the efficiency of such protection depends on the type of carotenoid. Rhodobacter sphaeroides synthesizes spheroidene as the main carotenoid under anaerobic conditions whereas, in the presence of oxygen, the enzyme spheroidene monooxygenase catalyses the incorporation of a keto group forming spheroidenone. We performed ultrafast transient absorption spectroscopy on membranes containing reaction center-light-harvesting 1-PufX (RC-LH1-PufX) complexes and showed that when oxygen is present the incorporation of the keto group into spheroidene, forming spheroidenone, reconfigures the energy transfer pathway in the LH1, but not the LH2, antenna. The spheroidene/spheroidenone transition acts as a molecular switch that is suggested to twist spheroidenone into an s-trans configuration increasing its conjugation length and lowering the energy of the lowest triplet state so it can act as an effective quencher of singlet oxygen. The other consequence of converting carotenoids in RC-LH1-PufX complexes is that S(2)/S(1)/triplet pathways for spheroidene is replaced with a new pathway for spheroidenone involving an activated intramolecular charge-transfer (ICT) state. This strategy for RC-LH1-PufX-spheroidenone complexes maintains the light-harvesting cross-section of the antenna by opening an active, ultrafast S(1)/ICT channel for energy transfer to LH1 Bchls while optimizing the triplet energy for singlet oxygen quenching. We propose that spheroidene/spheroidenone switching represents a simple and effective photoprotective mechanism of likely importance for phototrophic bacteria that encounter light and oxygen.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- bakteriochlorofyly chemie metabolismus MeSH
- buněčná membrána metabolismus MeSH
- karotenoidy chemie metabolismus MeSH
- kyslík metabolismus MeSH
- molekulární struktura MeSH
- přenos energie účinky záření MeSH
- Proteobacteria chemie metabolismus MeSH
- Rhodobacter sphaeroides chemie metabolismus MeSH
- spektrofotometrie MeSH
- světlo MeSH
- světlosběrné proteinové komplexy chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
