Visual proteomics of Mycoplasma pneumoniae with advanced photocrosslinking

• Applicants: Julia Mahamid, Ph.D.; Professor Dr. Juri Rappsilber
• Subject Area: Biochemistry ; Structural Biology
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 426290502

The project aims to advance the methodologies for studying protein interactions and structures in complex biological systems, with the potential for broader applications beyond Mycoplasma pneumoniae. To achieve this, the project will: (1) Enhance Crosslinking Mass Spectrometry (MS) techniques to better elucidate protein-protein interactions in M. pneumoniae. This involves developing and unifying existing analytical pipelines to handle photoactivatable crosslinks and improving membrane permeability of these crosslinkers. (2) Benchmark the applicability of the photoactivatable crosslinker SDA (succinimidyl 4,4’-azipentanoate) for proteome-wide crosslinking MS to achieve broader coverage of the M. pneumoniae proteome, improve the identification of transient protein interactions, and map complex protein networks at a higher spatial resolution compared to previous datasets. (3) Leverage high-throughput structural modelling based on the in-house developed AlphaLink software and crosslinking MS data to accelerate the discovery of protein complexes and structural biology insights. Specifically, it aims to characterize unresolved densities within the ribosome-associated Cage complex to understand its structure and function. (4) Explore the use of crosslinking MS-based models to enhance image analysis in cryo-ET data, with the goal of improving the identification and structural determination of protein complexes within the native cellular environment. (5) Validate high-resolution structural models using existing cryo-ET data and develop a pipeline for de novo determination of the cryo-ET structure of conserved and novel protein complexes in M. pneumoniae. The successful identification and structural modelling of these complexes will contribute to a comprehensive understanding of the cellular organization of this minimal genome bacterium.

• DFG Programme: Research Grants