Beck, Andreas. On the interaction of cationic compounds with ABC-transporters and lipid membranes. 2012, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_9885
Moreover we found that the ATPase activity of Sav1866 was not only stimulated by drugs, but also by an intrinsic cationic lipid, lysyl-DPPG. Our results indicated that Sav1866 may be involved in the transport of the cationic lipid to the outer leaflet of the membrane.
Beside the characterization of the ATPase activity of Sav1866 the reconstitution process was investigated. Reconstitution of membrane proteins is a semi-empirical task due to the lack of quantitative analysis of the process. Model membrane vesicles were therefore solubilized with a non-ionic detergent (C12E8). The detergent was removed with the help of Bio-Beads SM2 from Biorad. The vesicle size during the reconstitution process was directly measured by means of dynamic light scattering. Isothermal titrations calorimetry was used to quantify the degree of detergents removal. We found that still 9 mol% detergents per lipid were left after treatment.
Moreover the effect of a positively charged compound, dodecyltrimethylammonium chloride (DTAC), on neutral and negatively charged membranes was investigated in detail. The thermodynamic properties of DTAC were compared with the negatively charged counterpart sodium dodecyl sulphate (SDS). After correction for electrostatic contribution we found that SDS incorporates 10-15 times better into a membrane than DTAC, what is explained by the smaller headgroup of SDS. However SDS solubilizes the membrane at much lower critical ratios than DTAC. We revealed the movement of the POPC headgroup upon incorporation of the positively or the negatively charged detergent molecule by means of 2H-NMR. We found that upon incorproration of the cationic detergent the choline moiety of the lipid moves toward the plane of the bilayer, and vice versa for the anionic molecule. We conclude that the solubilization behaviour of the lipid membranes by charged detergnets is not only dependent on the structural properties of the detergents but also on the effective charge of the detergent molecule.
As a last point we investigated the critical role of TipF in assembly of the flagellum in Caulobacter crescentus. ITC experiemnts were performed to give insight into the binding properties of c-di-GMP with TipF. C-di-GMP is a second messenger involved in controlling growth and developpement in bacteriae. In vivo studies showed that TipF binds c-di-GMP, the binding constant was however unknown. We showed that c-di-GMP binds TipF with high affinity (Kd = 0.4 µM).
|Committee Members:||Seelig, Joachim and Klostermeier, Dagmar|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Biophysical Chemistry (Seelig A)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||167 S.|
|Last Modified:||30 Jun 2016 10:48|
|Deposited On:||08 May 2012 07:38|
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