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| Home > Publications database > Novel stealth carrier strategies for the structural investigation of ABC transporters by small-angle neutron scattering |
| Dissertation / PhD Thesis | PUBDB-2023-01405 |
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2023
Hamburg
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Please use a persistent id in citations: urn:nbn:de:gbv:18-ediss-107447 doi:10.3204/PUBDB-2023-01405
Abstract: MsbA is an essential, prokaryotic ATP binding cassette (ABC-)transporter which transportslipopolysaccharides, lipid A, and glycolipids from the inner to the outer leaflet of the bacterialinner membrane. Also, MsbA can function as a multidrug-resistance transporter and could betarget for new antibiotic developments and thus shows high biomedical relevance. However,the exact sequence of events during the transport mechanism is still not fully understood.In this dissertation, MsbA was incorporated into Saposin lipoprotein nanoparticles (Salipro)and was analysed in a single-particle cryo-electron microscopy (cryo-EM) experiment. ADP-vanadate (ADP-Vi) was used to trap MsbA in the post-hydrolysis state. In this occluded state,we improved the resolution to 3.5 Å, allowing us to model all side chains and visualise detailedinteractions of ADP-Vi and Mg2+ in the nucleotide-binding site. Furthermore, the analysis ofthe apo state of MsbA in Salipro indicates great flexibility of MsbA inside the lipid carrier withdifferent opening angles.Analysing small-angle X-ray scattering (SAXS) data of integral membrane proteins is oftenchallenging due to the scattering contribution of the stabilising system of the membrane protein(detergents, membrane scaffold protein (MSP)-based nanodiscs, Salipro etc.). With the help offractional deuterium labelled Salipro, the Salipro becomes neutron invisible by maximising theneutron scattering contribution of MsbA to simplify the small-angle neutron scattering (SANS)analysis. In the first step, a large-scale expression protocol of match-out deuterated Saposin A(dSapA) was developed using the high cell density culture (HCDC) in a fermenter to exchangehydrogen to deuterium of 70% of the non-exchangeable hydrogen atoms. Then, in a series ofcontrast variation small-angle neutron scattering (SANS) experiments, different componentsof the complex of MsbA in hydrogenated and partially deuterated Salipro were matched out,demonstrating the suitability of matching out components in the Salipro system. Next, theentire Salipro was matched out to measure SANS of MsbA in a stealth Salipro (dSapA withfractional deuterium labelled POPC) in 100% D2O. This stealth Salipro allows directobservation of the solubilised membrane protein without contribution from the surroundinglipid carrier. The match-out was confirmed via a comparison of the experimental SANS datato the calculated scattering of the previously described cryo-EM structure.In a time-resolved SAXS experiment initiated by stopped-flow mixing, the catalytic cycle ofMsbA incorporated into Salipro was investigated. The sample was mixed with ATP-Mg2+ andanalysed by SAXS over the first 1000 ms. Our data allowed us to structurally characteriseIImajor states of MsbA involved in the catalytic reaction with ATP. In addition, MsbAcontaining a catalytically inactive point mutation (MsbA-EQ) incorporated into Salipro andMSP-based nanodiscs were characterised in a time-resolved batch mode SAXS experiment.The ATP binding is not affected in both lipid carrier systems, while the speed of hydrolysisand the ADP/phosphate release are significantly reduced. Interestingly the radius of gyration(Rg) increases significantly above the starting Rg, which could be explained by a possible widerinward open state of MsbA compared to the apo conformation in Salipro/MSP-basednanodiscs.
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