@article{HeuvelingFrochauxZiomkowskaetal.2014,
  author    = {Heuveling, Johanna and Frochaux, Violette and Ziomkowska, Joanna and Wawrzinek, Robert and Wessig, Pablo and Herrmann, Andreas and Schneider, Erwin},
  title     = {Conformational changes of the bacterial type I ATP-binding cassette importer HisQMP(2) at distinct steps of the catalytic cycle},
  series = {Biochimica et biophysica acta : Biomembranes},
  volume    = {1838},
  journal   = {Biochimica et biophysica acta : Biomembranes},
  number    = {1},
  publisher = {Elsevier},
  address   = {Amsterdam},
  issn      = {0005-2736},
  doi       = {10.1016/j.bbamem.2013.08.024},
  pages     = {106 -- 116},
  year      = {2014},
  abstract  = {Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type land type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (1A0-Hi5QMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg2+ ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.},
  language  = {en}
}
@article{WessigWawrzinekMoellnitzetal.2011,
  author    = {Wessig, Pablo and Wawrzinek, Robert and Moellnitz, Kristian and Feldbusch, Elvira and Schilde, Uwe},
  title     = {A new class of fluorescent dyes based on 1,3-benzodioxole and [1,3]-dioxolo[4.5-f]benzodioxole},
  series = {Tetrahedron letters},
  volume    = {52},
  journal   = {Tetrahedron letters},
  number    = {46},
  publisher = {Elsevier},
  address   = {Oxford},
  issn      = {0040-4039},
  doi       = {10.1016/j.tetlet.2011.09.058},
  pages     = {6192 -- 6195},
  year      = {2011},
  abstract  = {We report on synthesis and photophysical properties of a new class of fluorescent dyes. They are characterized by large Stokes-shifts, long fluorescence lifetimes in organic solvents and a pronounced dependency of the fluorescence lifetime on the solvent polarity. Also worthy of note is the high bleaching stability. To provide access to biochemical and medical applications a series of derivatives were prepared, which exhibit specific reactivity towards different biologically relevant functional groups (carboxylic acids, amines, maleimides, N-hydroxysuccinimide esters). Furthermore, two alkynes were prepared, which could be used in 'Click' chemistry.},
  language  = {en}
}
@article{SagollaLoehmannsroebenHille2013,
  author    = {Sagolla, Kristina and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Hille, Carsten},
  title     = {Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells},
  series = {Analytical \& bioanalytical chemistry},
  volume    = {405},
  journal   = {Analytical \& bioanalytical chemistry},
  number    = {26},
  publisher = {Springer},
  address   = {Heidelberg},
  issn      = {1618-2642},
  doi       = {10.1007/s00216-013-7290-6},
  pages     = {8525 -- 8537},
  year      = {2013},
  abstract  = {Calcium (Ca2+) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca2+ concentration ([Ca2+](i)) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca2+](i) is the use of fluorescent Ca2+ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca2+ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca2+-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca2+-free and Ca2+-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca2+-free and Ca2+-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca2+ sensitivity is reduced. Nevertheless, quantitative [Ca2+](i) recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.},
  language  = {en}
}