TY - GEN A1 - Müller, Urs A1 - Weise, Frank A1 - Grell, S. ED - Fort, R. ED - de Buergo, R. T1 - The long term effects of a water repellent treatment on a volcanic tuff T2 - Heritage, Weathering and Conservation Conference (HWC 2006) CY - Madrid, Spanien DA - 2006-06-21 PY - 2006 SN - 0-415-41272-2 VL - 2 SP - 777 EP - 784 PB - Taylor & Francis CY - London AN - OPUS4-12509 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Marinelli, F. A1 - Kuhlmann, S. I. A1 - Grell, E. A1 - Kunte, Hans-Jörg A1 - Ziegler, C. M. A1 - Faraldo-Gómez, J. D. T1 - Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins N2 - Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding Proteins (SBP) have a strong affinity for their ligands; yet, Substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of APO TeaA, the SBP of the Na!-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581T, and compare it with the substrate-bound structure previously reported. Conformational freeenergy landscape calculations based upon molecular Dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their Membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former. KW - Binding thermodynamics KW - Periplasmic binding protein KW - Secondary transporter KW - ABC transporter KW - Replica-exchange metadynamics PY - 2011 U6 - https://doi.org/10.1073/pnas.1112534108 SN - 0027-8424 SN - 1091-6490 VL - 108 IS - 49 SP - E1285 EP - E1292 AN - OPUS4-37681 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Marinelli, F. A1 - Kuhlmann, S.I. A1 - Grell, E. A1 - Kunte, Hans-Jörg A1 - Ziegler, C. M. A1 - Faraldo-Gómez, J.D. T1 - Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins N2 - Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na+-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581T, and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former. KW - Binding thermodynamics KW - Periplasmic binding protein KW - Secondary transporter KW - ABC transporter KW - Replica-exchange metadynamics PY - 2011 U6 - https://doi.org/10.1073/pnas.1112534108 SN - 0027-8424 SN - 1091-6490 VL - 108 IS - 49 SP - E1285 EP - E1292 PB - National Academy of Sciences CY - Washington, DC AN - OPUS4-25049 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -