Česká společnost pro hmotnostní spektrometrii

(CEEPC/IPM/CMSC 2022 - ThS-07)
Overlooked oligomerization process in Azurin, a model metalloprotein

Roman Tuzhilkin ¹, Miroslav Šulc ^1 *

1. Department of Biochemistry, Faculty of Science, Charles University

Abstrakt

Pseudomonas aeruginosa azurin is a small blue copper protein commonly used as a model in electron transfer (ET) experiments due to its characteristic physiological function and UV-VIS spectrum changes during ET activity. Its lesser-known characteristic is formation of oligomers which can influence ET in solution even under concentrations close to physiological. In this study we have employed chemical cross-linking of different azurin forms with lysine-reactive chemicals (DSS or DSG) paired with SDS-PAGE visualisation and MS identification to map the interactions in aforementioned oligomers. Additionally, we have employed a novel purification approach to prepare apo-azurin – a protein deprived of the central copper ion which is crucial both structurally and functionally. The same approach was also applied to prepare zinc metallised form to substitute the central copper ion. Acquired data correlates with our previous results and shows important role of C-terminal β-sheet in the interaction. Based on MS data and site directed mutagenesis, we predict the cross-link is formed between K122 residues of two azurin monomer units. The mutant protein with K122 substitution produces no oligomers covalently linked by DSS or DSG on SDS-PAGE, even though other experiments revealed generally higher oligomerization rate for this mutant. More than that, the nature of the metal cofactor or its absence influences DSS/DSG visualisation of azurin oligomerization through altering exposure of C-terminal β-sheet (Cu > Zn > Apo). To sum up, we have resolved the structure of largely overlooked azurin oligomers and uncovered abnormal oligomerization behaviour occurring in non-conventional azurin metal forms.

* Korespondující autor: mis@natur.cuni.cz

Poděkování:

The project was supported by Charles University (GAUK n. 1538119) and Czech Science Foundation (20-28126S).