Identifying and characterizing µ-membrane proteins in prokaryotic terminal oxidases and proteomes
Max-Planck-Institute of Biophysics, Frankfurt (Germany), Department of Molecular Membrane Biology
Julian Langer and Hartmut Michel
Our work in this SPP has two major aims: In the first part, we plan to gain new insights into the roles of µ-membrane proteins in prokaryotic terminal oxidases. In the second part, we want to perform membrane proteomics screening experiments to identify membrane-embedded small proteins in selected model organisms.
- We plan to investigate the functional roles of the oxidase µ-membrane protein subunits recently identified by our groups. Further, we want to examine how common µ-subunits are in oxidase complexes from different organisms. To this end, we will purify different oxidase types from coli, G. thermodenitrificans, P. stutzeri, M. phlei and other prokaryotes, and make use of our MALDI-MS/MS setup to directly sequence and identify small proteins. We will then generate genetic deletion variants of the identified small proteins to investigate possible roles in complex assembly and oxidase activity. Using homologously, recombinantly produced small proteins, we will map their interaction partners to gain further insights into their physiological roles. We will also perform functional studies, e.g. activity measurements, on the oxidase complexes in the presence and absence of the small proteins to further elucidate their functional roles.
- In addition to the targeted studies on oxidase complexes, we plan to perform membrane µ-proteome screening experiments in stutzeri, M. phlei and G. thermodenitrificans. Initially, we will screen the genomes of these model organisms to identify possible ORFs for µ-membrane proteins. Then we attempt to extract and sequence the respective membrane µ-proteomes directly using a prototype Bruker Rapiflex MALDI-TOF/TOF tandem mass spectrometer.
Project related publications:
- Alvarez-Castelao B, Schanzenbächer CT, Hanus C, Glock C, Tom Dieck S, Dörrbaum AR, Bartnik I, Nassim-Assir B, Ciirdaeva E, Mueller A, Dieterich DC, Tirrell DA, Langer JD, Schuman EM. Cell-type-specific metabolic labeling of nascent proteomes in vivo. Nat Biotechnol. 2017.
- Eisinger ML, Dörrbaum AR, Michel H*, Padan E*, Langer JD*. Ligand-induced conformational dynamics of the Escherichia coli Na(+)/H(+) antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry. Proc Natl Acad Sci U S A. 2017.
- Bausewein T, Mills DJ, Langer JD, Nitschke B, Nussberger S, Kühlbrandt W. Cryo-EM Structure of the TOM Core Complex from Neurospora crassa. Cell. 2017,
- Kohlstaedt M, Buschmann S, Langer JD, Xie H, Michel H*. Subunit CcoQ is involved in the assembly of the Cbb(3)-type cytochrome c oxidases from Pseudomonas stutzeri ZoBell but not required for their activity. Biochim Biophys Acta. 2017.
- Demmer JK, Rupprecht F, Eisinger ML, Ermler U, Langer JD*. Ligand binding and conformational dynamics in a flavin-based electron bifurcating enzyme complex revealed by HDX-MS. FEBS Letters. 2016.
- Schanzenbächer C, Sambandan S, Langer JD*, Schuman EM*. Nascent Proteome Remodeling following Homeostatic Scaling at Hippocampal Synapses. Neuron. 2016.
- Safarian S, Rajendran C, Müller H, Preu J, Langer JD, Ovchinnikov S, Hirose T, Kusumoto T, Sakamoto J, Michel H*. Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases. Science. 2016.
- Kohlstaedt M, Buschmann S, Xie H, Resemann A, Warkentin E, Langer JD*, Michel H*. Identification and Characterization of the Novel Subunit CcoM in the cbb3 Cytochrome c Oxidase from Pseudomonas stutzeri ZoBell. MBio. 2016.
- Preiss L, Langer JD, Yildiz Ö, Eckhardt-Strelau L, Guillemont JE, Koul A, Meier T*. Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline. Sci Adv. 2015.
- Xie H, Buschmann S, Langer JD, Ludwig B, Michel H*. Biochemical and biophysical characterization of the two isoforms of cbb3-type cytochrome c oxidase from Pseudomonas stutzeri. J Bacteriol. 2014
- Pogoryelov D, Klyszejko AL, Krasnoselska GO, Heller EM, Leone V, Langer JD, Vonck J, Müller DJ, Faraldo-Gómez JD, Meier T*. Engineering rotor ring stoichiometries in the ATP synthase. Proc Natl Acad Sci U S A. 2012.
- Pogoryelov D, Krah A, Langer JD, Yildiz Ö, Faraldo-Gómez JD, Meier T*. Microscopic rotary mechanism of ion translocation in the Fo complex of ATP synthases. Nat Chem Biol. 2010.
- Buschmann S, Warkentin E, Xie H, Langer JD, Ermler U, Michel H*. The structure of cbb3 cytochrome c oxidase provides insights into proton pumping. Science. 2010.
- Iwata S, Ostermeier C, Ludwig B, Michel H*. Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature. 1995.