Showing papers by "Nina Morgner published in 2013"

Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump

Abstract: Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent KD values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.

Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation

Abstract: F-type ATPases are highly conserved enzymes used primarily for the synthesis of ATP. Here we apply mass spectrometry to the F1FO-ATPase, isolated from spinach chloroplasts, and uncover multiple modifications in soluble and membrane subunits. Mass spectra of the intact ATPase define a stable lipid 'plug' in the FO complex and reveal the stoichiometry of nucleotide binding in the F1 head. Comparing complexes formed in solution from an untreated ATPase with one incubated with a phosphatase reveals that the dephosphorylated enzyme has reduced nucleotide occupancy and decreased stability. By contrasting chemical cross-linking of untreated and dephosphorylated forms we show that cross-links are retained between the head and base, but are significantly reduced in the head, stators and stalk. Conformational changes at the catalytic interface, evidenced by changes in cross-linking, provide a rationale for reduced nucleotide occupancy and highlight a role for phosphorylation in regulating nucleotide binding and stability of the chloroplast ATPase.

Compositional and Structural Analyses of the Photosystem II Isolated from the Red Alga Cyanidioschyzon Merolae

Abstract: Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extremely low pH levels and moderately high temperatures. The photosynthetic apparatus of the red alga Cyanidioschyzon merolae represents an intermediate type between cyanobacteria and higher plants, suggesting that this alga may provide the evolutionary link between prokaryotic and eukaryotic phototrophs. While red algal PSI resembles that of the higher plants, the PSII complex is more reminiscent of the cyanobacterial ancestor in that it contains phycobilisomes as the light-harvesting system instead of Chla/b binding proteins of green algae and higher plants, as well as the PsbU and PsbV subunits stabilising the oxygen evolving complex (OEC). The most remarkable feature of the red algal PSII is the presence of the fourth extrinsic protein of 20 kDa (PsbQ’) which is not found in the cyanobacterial OEC and which is distantly related with the green algal PsbQ. This feature together with some differences in the structural cooperation between the OEC subunits suggests that the lumenal side of red algal PSII may vary from the prokaryotic ancestor. In order to elucidate the structural differences between cyanobacterial and eukaryotic PSII, we have isolated highly active and stable dimeric complexes of the C. merolae PSII and subjected them to high throughput crystallization and mass spectrometry analyses. Here we report the full subunit composition and preliminary results of 3D crystallization of the dimeric C. merolae PSII.