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Principal Investigator

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P24  Nina Morgner
Juniorprofessor

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Institute of Physical and
Theoretical Chemistry
Goethe University Frankfurt a.M.
Max-von-Laue-Str. 7
60438 Frankfurt am Main, Germany

Phone +49 (0)69 798-29441

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P24 Dynamics of complex membrane assemblies probed by native mass spectrometry


Many essential cell functions depend on the interaction of proteins embedded in the cell membranes. To capture the dynamic of these processes methods are required that allow monitoring of the response of membrane protein complexes to stimuli such as binding / hydrolysis of nucleotides, specific interactions with cofactors, substrates, lipids, or post-translational modifications.

We plan to investigate the dynamic processes of several complexes involved in cross membrane transport, such as the ABC transporter TmrAB, the P-type ATPase KdpFABC an unusual chimera between ion channels and ion pumps. Other examples are the translocon complex of outer chloroplast membranes (TOC) or the macromolecular peptide loading complex (PLC). Using strategic mutants or trapping of different cycle stages by incubation with inhibitors will allow examination of these systems throughout their catalytic cycles. We will employ LILBID (Laser Induced Liquid Bead Ion Desorption) and nESI (nano Electrospray Ionization) to monitor subunit stoichiometries and the complexes dependence on associated lipids, substrates, or nucleotide binding/hydrolysis including conformational changes via mass spectrometry (MS) and ion mobility (IM).

For many processes in the cell membrane the non-covalent assembly of membrane protein complexes is essential. Nevertheless the driving forces for the complex assemblies are difficult to determine and to predict, therefore new methods are needed to investigate these. While the binding energy of a protein and a ligand or even two proteins can be determined when both binding partners are available separately (e.g. Isothermal Titration Calorimetry (ITC) or Surface Plasmon Resonance (SPR)), the experimental determination of binding energies between proteins of an intact protein complex is much more challenging. Especially for membrane proteins even established methods (ITC) do not work reliably. We plan to develop a LILBID based approach to determine binding energies of non-covalently bound membrane protein complexes in a quantitative way, depending on the LILBID laser and desorption process. We will establish this method using a set of membrane protein/nanobody complexes, with known binding affinities and then extend it to larger membrane protein complexes in collaboration with several groups within the consortium.

 

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LILBID-MS spectra of A. woodii c-rings taken in different instrument modes, showing the intact c-ring (A) or a series of subcomplexes (B/C), which reveal the stoichiometry of the c-subunits c2/3 (2 transmembrane helices (TMH)) and c1 (4 TMH)  to be 9:1.
The finding of this fixed composition could exclude the possibility of different A.woodii ATPase populations with varying c-ring stoichiometries, which up to then were only known to have 22 TMH, as observed by Electron microscopy of 2D crystals from A. woodii c rings (D) [modified after Fritz et al, FEBS Journal 275, 1999-2007, (2008)].  The softness of the method is determined by the intensity of the desorption laser which irradiates the sample droplet, generated by an on-demand droplet generator (E).

 

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Publications

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Maciejko J, Mehler M, Kaur J, Lieblein T, Morgner N, Ouari O, Tordo P, Becker-Baldus J, Glaubitz C (2015) Visualizing specific cross-protomer interactions in the homo-oligomeric membrane protein proteorhodopsin by dynamic-nuclear-polarization-enhanced solid-state NMR. J Am Chem Soc 137, 9032-43.

Bechara C, Nöll A, Morgner N, Degiacomi, MT, Tampé R*, Robinson CV* (2015) A subset of annular lipids is linked to the flippase activity of an ABC transporter. Nature Chemistry 7, 255-62.


Schmidt, C., Zhou, M., Marriott, H., Morgner, N., Politis, A. and Robinson, C.V. (2013) Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation. Nature Commun 4, 1985. 


Marcoux, J., Wang, S.C., Politis, A., Reading, E., Ma. J., Biggin, P.C., Zhou, M., Tao, H., Zhang, Q, Chang, G., Morgner*, N. and Robinson+, C.V. (2013) Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc Natl Acad Sci USA 110(24), 9704-9709.

Zhou#, M., Morgner#, N., Barrera#, N.P., Politis, A., Isaacson, S.C., Matak Vinkovic, D., Murata, T., Bernal, R.A., Stock, D. and Robinson, C.V. (2011) Intact V-type ATPases survive in the gas phase revealing specific lipid binding in the transmembrane sector. Science 334 (6054), 380-385.

Ebong I-O#, Morgner N#, Zhou M, Saraiv MA, Daturpalli S, Jackson S, Robinson CV (2011) Heterogeneity and dynamics in the assembly of the Heat Shock Protein 90 chaperone complexes. Proc Natl Acad Sci USA 108, 17939-44. 

Vonck, J., Pisa, K., Morgner, N., Brutschy, B. and Müller, V. (2009) Three-dimensional structure of A1A0 ATPsynthase from the hyperthermophilic archaeon Pyrococcus furiosus by electron microscopy. J Biol Chem 284(15), 10110-10119.

Fritz#, M., Klyszejko#, A.L., Morgner#, N., Vonck, J., Brutschy, B., Muller, D.J., Meier, T. and Müller, V. (2008) An intermediate step in the evolution of ATPases: a hybrid FO-VO rotor in a bacterial Na+ F1FO ATP synthase. FEBS J 275, 1999-2007.

Morgner, N., Zickermann, V., Kerscher, S., Wittig, I., Abdrakhmanova, A., Barth, H.-D., Brutschy, B. and Brandt, U. (2008) Subunit mass fingerprinting of mitochondrial
complex I. Biochim Biophys Acta 1777, 1384-1391.

Meier, T., Morgner, N., Matthies, D., Pogoryelov, D., Keis, S., Cook, G.M., Dimroth, P. and Brutschy, B. (2007) A tridecameric c ring of the adenosine triphosphate (ATP) synthase from the thermoalkaliphilic Bacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemical proton potential. Mol Microbiol 65(5), 1181-1192.

Morgner, N., Kleinschroth, T., Barth, H.-D., Ludwig, B. and Brutschy, B. (2007) A novel approach to analyze membrane proteins by laser mass spectrometry: from protein subunits to the integral complex. J Am Soc Mass Spectrom 18, 1429-1438. 

*  joint corresponding authors
# denotes equal contribution

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Collaborations

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Glaubitz (P06), Geertsma (P23), Tampé (P16), Hänelt (P22), Schleiff (P17), 
Dötsch/Bernhard (P02), Abele (P09), Müller (P14), Ernst (P21)