Characterization of structure and function of small heat shock like proteins from psychrophilic yeast, glaciozyma antarctica pi12 in response to thermal stress
Antarctica, with its unique geography and extreme climate, serves as the primary habitat for bacteria. Among these microorganisms, Antarctic subglacial species have developed the ability to endure high pressure and severe cold conditions. Research has shown that molecular chaperones play a crucial r...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2023
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| Subjects: | |
| Online Access: | https://eprints.ums.edu.my/id/eprint/40549/1/24%20PAGE.pdf https://eprints.ums.edu.my/id/eprint/40549/2/FULLTEXT.pdf |
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| Summary: | Antarctica, with its unique geography and extreme climate, serves as the primary habitat for bacteria. Among these microorganisms, Antarctic subglacial species have developed the ability to endure high pressure and severe cold conditions. Research has shown that molecular chaperones play a crucial role in preventing protein degradation and facilitating protein refolding under heat stress. Specifically, small heat shock like-proteins (sHSPs) have been found to interact with partially unfolded proteins prone to aggregation, thereby reducing cellular damage. The exceptional functionality of psychrophilic sHSPs at low temperatures presents an opportunity to explore the relationship between protein structure, stability, flexibility, and dynamic conformation. This study aims to investigate the role of sHSPs derived from Glaciozyma antartica and examine the connection between their molecular structure and heat adaptation. Out of the four sHSP genes identified in G. antarctica, two namely GasHSP07-010 and GasHSP12-338, were amplified and cloned using E. coli BL21(DE3). The proteins encoded by these genes were expressed at 37°C overnight and subsequently purified using immobilized metal chelate affinity chromatography (IMAC). The purified proteins underwent both a citrate synthase assay and a thermotolerance assay. Furthermore, comparative modeling of these genes was performed using CHIMERA, aligning them against the Homo sapiens (2YRT) and Schizosaccharomyces pombe (3W1Z) strains. The quality of the modeled structures was evaluated using the Ramachandran plot, errat, and verify3D. Results from the in vitro thermotolerance assay demonstrated that GasHSP07-010 and GasHSP12-338 protected E. coli cells from lethal temperatures of 55°C for up to 30 and 60 minutes, respectively. An aggregation assay using citrate synthase (CS) further revealed the chaperone activity of both sHSPs, as they effectively protected CS from complete aggregation. The sHSP:CS at a ratio of 2:1 was found to be more effective than the 1:1 ratio for both G. antarctica sHSP proteins. The 2:1 ratio might have functioned better than the 1:1 ratio because sHSP requires a specific ratio of protein concentration and non-native protein to generate stable and effective complexes. Additionally, real-time PCR analysis showed that gashsp12-338 expression increased by 1.38-fold under high heat stress and 2.33- fold under cold stress compared to the control temperature of 12°C. As a result of exposure to the fatal temperature of 20°C, both gashsp07-010 and gashsp12-338 expression levels were downregulated. Interestingly, at 30°C, both gashsp07-010 and gashsp12-338 levels were upregulated 2-fold compared to the expression at 20°C. It was possible that at 30°C, the presence of non-native proteins such as aggregates at a certain level triggered the expression of both sHSP. These findings reflect the diverse function of sHSP in G. antarctica that may play different roles in thermal adaptation. Comparative modeling of G. antarctica sHSP structures uncovered noteworthy alterations in the amino acid composition. In the tertiary structure of GasHSP07-010, an amino acid transition from non-charge to polar resulted in reduced interactions and increased stability. Conversely, GasHSP12-338 exhibited an amino acid change to a non-polar form, leading to diminished amino acid interactions and enhanced structural stability. These modifications loosen the strong ionic interactions and create a flexible connection which allows conformation change in the protein structures similar to the cold-adapted proteins in hypersaline conditions which play an important role in protein solubility and flexibility to increase the speed of enzymatic bindings and reactions. These structural adaptations likely contribute to the flexibility and stability required for the functional activity of these proteins at low temperatures and their ability to protect other proteins during heat stress. The findings of this study shed light on the thermal protection mechanisms employed by sHSPs and offer valuable insights into their functionality. |
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