STRUCTURAL PROTEOMICS OF FTSH COMPLEXES USING CROSS-LINKING MASS SPECTROMETRY

Date

2023-8

Author

Akkulak, Hatice

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FtsH proteases are responsible for membrane protein quality control and are highly conserved in bacteria, mitochondria, and chloroplasts. FtsH contains an ATP- dependent endonuclease that degrades improperly folded integral membrane proteins and interacts with other membrane proteins HflK and HflC, modulating this process. This thesis investigated the composition and structure of FtsH complexes using cross-linking mass spectrometry (XL-MS) based structural proteomics. Recombinant expression of protein complexes was performed in E. coli, inner membranes were solubilized, and proteins were purified using affinity chromatography. The residues of proteins in close proximity were covalently bound to each other via cross-linkers. Cross-linked proteins were then enzymatically digested and analyzed by the high-resolution MS. Commonly used XL-MS software tools, MaxLynx, MeroX, MS Annika, and XlinkX were assessed to elucidate the protein interactions within the membrane protein complexes containing FtsH, HflK, and HflC. The cross-link search analysis yielded over 300 inter- and intra-protein interactions. The results provide valuable information regarding the complex structure and function and could be applied to molecular docking for structural modeling.

Subject Keywords

Cross-linking Mass Spectrometry, Protein Interactions, Structural Proteomics, FtsH Complexes, Membrane Proteins

URI

https://hdl.handle.net/11511/105248

Collections

Graduate School of Natural and Applied Sciences, Thesis