Analysis of the structure and function of CLIC4 in aqueous and membrane environments

Zhu, Junjie (2025) Analysis of the structure and function of CLIC4 in aqueous and membrane environments. PhD thesis, University of Glasgow.

Abstract

CLIC4 is a member of the CLIC family of soluble proteins that are capable of spontaneous incorporation into lipid intracellular membranes to form chloride selective ion channels. Given the critical functions of the biological plasma membranes, artificial lipid-based membranes mimicking the natural membranes have been used in characterizing membrane proteins in a native-like environment. Here in this study, His-tagged CLIC4 protein expression was optimized by testing in vivo and in vitro expression systems. Furthermore, the conformational structure of CLIC4 protein was examined in aqueous and in an artificial liposome membrane environment in presence and absence of oxidizing agent. Where, under non-reducing aqueous environment, CLIC4 tends to oligomerize upon incubation at 37oC in a time-dependent manner compared to the stable CLIC4 monomers at 4oC. In artificial liposome membrane environment, CLIC4 oligomerization appeared to be independent of the oxidizing environment. Given that, cysteine (Cys) residues in other CLIC family members have been implicated in the protein conformational structure, we attempted to investigate the impact of CLIC4 Cys residues on the CLIC4 conformational structure within the membrane liposomes. Indeed, PEGylation analysis of CLIC4 mutants, where Cys 35, Cys 100, Cys 189 and Cys 234 were substituted with serine indicates enhanced solvent accessibility at C35S and C189S compared C100S or C234S CLIC4 mutants, thus highlighting the impact of Cys 35 and Cys 189 on CLIC4 PEGylation. Furthermore, analysis of truncated CLIC4 protein fragments including 100, 150, 220 and 270 amino acids suggest that Cys 35 limits the solvent accessibility to CLIC4 protein compared to Cys 100, Cys 189 and Cys 234. However, the solvent accessibility to Cys 100 and Cys 189 were not impacted by Cys 35. Interestingly, in presence of Cys 35, Cys 234 appeared to negatively impact the solvent accessibility to Cys 100 and Cys 189. Taken together, these results highlight the critical role of Cys residues on the CLIC4 protein conformational structure and incorporation into membranes and further suggest that Cys 35 and Cys 234 could be target sites for further CLIC4 modification to induce targeted conformational changes.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from the Institute of Molecular, Cell and Systems Biology at the University of Glasgow, and the China Scholarship Council. Due to copyright issues this thesis is not available for viewing.
• Subjects: Q Science > QH Natural history > QH301 Biology
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Funder's Name: China Scholarship Council (CSC)
• Supervisor's Name: Woolhead, Professor Cheryl
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85200
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 Jun 2025 11:05
• Last Modified: 17 Jun 2025 13:04
• Thesis DOI: 10.5525/gla.thesis.85200
• URI: https://theses.gla.ac.uk/id/eprint/85200