TY - JOUR A1 - Heuveling, Johanna A1 - Frochaux, Violette A1 - Ziomkowska, Joanna A1 - Wawrzinek, Robert A1 - Wessig, Pablo A1 - Herrmann, Andreas A1 - Schneider, Erwin T1 - Conformational changes of the bacterial type I ATP-binding cassette importer HisQMP(2) at distinct steps of the catalytic cycle JF - Biochimica et biophysica acta : Biomembranes N2 - 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. KW - ABC transporter KW - Type I importer KW - Histidine transport KW - Limited proteolysis KW - Fluorescence lifetime KW - Altemate access model Y1 - 2014 U6 - https://doi.org/10.1016/j.bbamem.2013.08.024 SN - 0005-2736 SN - 0006-3002 VL - 1838 IS - 1 SP - 106 EP - 116 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Wessig, Pablo A1 - Wawrzinek, Robert A1 - Moellnitz, Kristian A1 - Feldbusch, Elvira A1 - Schilde, Uwe T1 - A new class of fluorescent dyes based on 1,3-benzodioxole and [1,3]-dioxolo[4.5-f]benzodioxole JF - Tetrahedron letters N2 - 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. KW - Fluorescent dyes KW - Fluorescence lifetime KW - Large Stokes-shifts KW - Heterocycles Y1 - 2011 U6 - https://doi.org/10.1016/j.tetlet.2011.09.058 SN - 0040-4039 VL - 52 IS - 46 SP - 6192 EP - 6195 PB - Elsevier CY - Oxford ER -