Identification and functional analyses of small membrane proteins in terminal oxidases and selected model organism proteomes

• Applicant: Dr. Julian Langer
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Biochemistry ; Biophysics
• Term: since 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 379643633

The work we propose for the second phase of SPP2002 is divided up in two parts: In the first part, we plan to continue and expand our analyses of small subunits in terminal oxidases, make use of HDX-MS to map peptide ligand binding sites and analyse conformational dynamics with collaboration partners in the SPP. In the second part, we will further develop our methodological approach and perform explorative analyses on small membrane proteins in selected model organisms.1) Identification of novel subunits in terminal oxidases and targeted functional studies. Terminal oxidases comprise essential elements of the respiratory chain and represent promising antimicrobial drug targets as they often have unique architectures distinct from their mammalian homologs. In our project we will expand our analyses of small subunits of terminal oxidases and screen different oxidases in selected model organisms including E. coli, P. stutzeri and Mycobacteria strains. Here our Rapiflex MALDI-TOF/TOF setup will be used to directly sequence and identify small subunits in purified protein complexes. A particular focus will be on isofom 2 of the E. coli bd oxidase, which we already successfully purified in our lab.Further we will expand our functional characterization of small subunits in terminal oxidases. We will focus on interactomics and functional studies on CydH/Ynhf in the bd oxidase (isoform 1) in E. coli, which we identified in the first round of SPP2002. To understand the role of the small subunits in oxidase assembly and activity, we will generate genetic deletion variants and conditional knock-out strains. We are also going to express tagged variants of the small subunits or raise specific antibodies or nanobodies against the native proteins for interactomics studies. In addition crosslinking based interactomics studies will be performed.Finally we will make use of HDX-MS to characterize ligand binding and conformational dynamics of terminal oxidases, both in the presence and absence of small subunits. We will also engage in multiple collaborations within this SPP to map peptide ligand binding sites in target protein complexes, and study the associated conformational changes. 2) Method development and explorative studies in selected prokaryote model systems. We will expand our explorative studies on functional roles of small polypeptides in different prokaryotes. This will include further method development and the application of these approaches to selected target organisms. We will improve our top-down, digest-free approach using ion mobility-coupled mass spectrometry to enrich for and improve detection of large peptides with low charge states.We will make use of our pipeline to screen and analyze E. coli for small membrane proteins, and perform proteomics studies of different P. stutzeri and E. coli knock-out strains of small proteins, including targets identified in the first round of the Priority program, under different conditions.

• DFG Programme: Priority Programmes
• Subproject of: SPP 2002: Small Proteins in Prokaryotes, an Unexplored World