Analysis of cyanobacterial protein complexes by advanced techniques of mass spectrometry

Mgr. Peter KONÍK

Doctoral thesis

Abstract:

For the last two decades, mass spectrometry (MS) is a method of choice for the identification of proteins involved in the formation of photosynthetic complexes in cyanobacteria. The aim of this thesis was to optimize and utilize MS methods beyond a simple protein identification for the model cyanobacterium Synechocystis sp. PCC 6803. Specifically, I focused on label free protein quantification, both in cell extracts and in isolated proteins, and structural proteomics by crosslinking (XL-MS). To this thesis I however included also my publications where I contributed by identification of proteins, that were co-isolated with tagged Photosystem II assembly factors CyanoP, Psb27, Psb28, Psb29 and Pam68. This research significantly broadened the knowledge about PSII biogenesis and connected the Psb29 protein with formation of heterocomplexes of FtsH proteases. In order to identify substrates of Synechocystis FtsH4 protease, I employed quantitative proteomic analysis of proteins co-eluted with catalytically-inactivated FtsH4trap enzyme. This work led to discovery of several Photosystem I assembly factors and Photosystem I-related protein such is IsiA, as FtsH4 substrates. Other group of identified proteins (substrates) seems to be specifically involved in CO2 uptake. Quantitative proteomics was further used to clarify the role of High-light inducible proteins (Hlips). A detail analysis of HliA and HliB pulldowns prepared from wild type and hliC backgrounds mapped protein-protein interactions involved in photoprotection of Photosystem II biogenesis. Utilizing XL-MS, I probed the structure of the ArgD-Gun4 protein complex that regulatory connects nitrogen metabolism and the biosynthesis of tetrapyrroles. The formation of the ArgD-Gun4 complex is promoted by ornithine, an intermediate of arginine biosynthesis.

Language used: English

Date on which the thesis was submitted / produced: 8. 1. 2024

Thesis defence

• Supervisor: doc. Ing. Roman Sobotka, Ph.D.

UNIVERSITY OF SOUTH BOHEMIA IN ČESKÉ BUDĚJOVICE

Faculty of Science

Doctoral programme / field:
Molecular and Cell Biology / Molecular and Cell Biology and Genetics