Advanced Photon Counting. Springer Series on FLuorescence , Springer, Benda, A. Fluorescence spectral correlation spectroscopy FSCS for probes with highly overlapping emission spectra. Express, 22, 3, The fast polarization modulation based dual-focus fluorescence correlation spectroscopy. Express, 22, 1, The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicing. Raster image correlation spectroscopy as a novel tool to study interactions of macromolecules with nanofiber scaffolds.
Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement. Understanding the molecular basis of enzyme-substrate interactions that contribute to enantioselectivity is important for constructing selective enzymes by protein engineering. An emerging group of enzymes that is explored for enatioselectivity are dehalogenases.
Haloalkane dehalogenases HLDs can convert a broad range of halogenated aliphatic substrates to their corresponding alcohols, and because of the simplicity of the reaction represent a good model system to study structural basis of reactivity and enantioselectivity. We showed that fluorescent solvent relaxation technique in synergy with MD simulations allows the characterization of hydration and dynamics at the entry to the active site pocket.
Our very recent experiments strongly indicate that these two parameters are linked to the enantioslectivity of certain designed dehalogenase enzymes. As this finding is important for the overall concept of protein design, we will expand this unique approach, which is based on the combination of the solvent relaxation technique, MD simulations, as well as protein design and characterization, to other well selected representative of this protein class with the aim to make more general conclusions which will be essential for the entire concept of de novo protein design.
Additionally, the fluorescent unnatural aminoacid UAA based on the coumarin can be inserted in the specific manner into the enzyme. By means of the steady state spectroscopy the hydration can also be determined.
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The gained results fully support the conclusions drawn from the above described techniques of solvent relaxation and MD simulations. In future we also plan to study gating mechanisms that help to control the ligand transport in certain HLDs.
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Specifically, a flexible tryptophan positioned at the tunnel mouth of some HLDs acts as a gate. We will monitor the transition between the open and close conformations of this gating residue by single molecule fluorescence. The coupling between the electron-transfer and structural relaxation dynamics is monitored by means of time-dependent fluorescence shifts and compared with the time resolved infrared spectroscopy in azurins and cytochromes using both the organic and complex inorganic chromophores.
The samples were prepared as droplets of the purified fluorescent proteins dissolved in phosphate buffered saline PBS, pH 7. The average laser power reaching the sample was precisely tuned with a polarizing attenuator consisting of a half-wave plate and a polarizing cube installed in front of microscope coupling dielectric filter. The SF10 prism compressor was used to compensate for the group velocity dispersion in the objective lens and other optical elements.
Advanced Fluorescence Reporters in Chemistry and Biology II
Fluorescence was excited by two-photon absorption of femtosecond laser, passed back through the objective lens and laser coupling filter and then was directed to the input of Acton SPi monochromator with two separate outputs. Average input laser power was 1.
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To precisely adjust irradiation intensity continuously variable neutral density filters were used. Same medium was used for imaging. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Chudakov, D. Fluorescent proteins and their applications in imaging living cells and tissues. Shaner, N. A guide to choosing fluorescent proteins.
Kremers, G. Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy. Berezin, M. Fluorescence lifetime measurements and biological imaging. Sarkisyan, K. Goedhart, J. Tryptophan-based chromophore in fluorescent proteins can be anionic. Bindels, D.
Advanced Fluorescence Reporters in Chemistry and Biology I | SpringerLink
Matela, G. Piatkevich, K. Extended Stokes shift in fluorescent proteins: chromophore-protein interactions in a near-infrared TagRFP variant. Ganesan, S. Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells. Biochemistry 46 , — Bogdanov, A. Suhling, K. Imaging the environment of green fluorescent protein. Nakabayashi, T.
Sensing of intracellular environments by fluorescence lifetime imaging of exogenous fluorophores. McLoskey, D. Desvergne, E. Perez-Inestrosa, H. Bouas-Laurent, G. Jonusauskas, J. Fabbrizzi, M. Licchelli, A. Poggi, G.
Rabaioli, A. Klimant, C. Huber, G. Liebsch, G. Neurauter, A.
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Stangelmayer, O. Two-Photon Fluorescence Fluctuation Spectroscopy. Chen, J.