Elucidation of structure and function of conserved hypothetical proteins related to thermal stress response in pedobacter cryoconitis BGs and glaciozyma antarctica PI12

Makdi Masnoddin (2022) Elucidation of structure and function of conserved hypothetical proteins related to thermal stress response in pedobacter cryoconitis BGs and glaciozyma antarctica PI12. Post-Doctoral thesis, Universiti Malaysia Sabah.

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Abstract

The genome data of two native Antarctic microbes, Pedobacter cryoconitis (bacterium) and Glaciozyma antarctica (yeast), highlighted the presence of specific proteins with unique adaptive features. However, most of these proteins were designated as conserved hypothetical proteins (HPs), complicating efforts to understand their cellular functions. Consequently, we aim to identify the structural features of the conserved HPs that were ideal for their function in response to temperature stress. We posit that proteins that respond to thermal stress may have unique structural flexibility and stability that allows them to function under thermal stress, protecting host organisms against cold denaturation and heat aggregation. To address, an in silica analysis of HPs was conducted, followed by an in vitro approach whereby all selected HPs were cloned, expressed in Escherichia coli, purified, and subjected to crystal screening experiment. Purified recombinant proteins were assessed via colorimetric ATPase and citrate synthase aggregation assays and quantitative PCR ( qPCR) gene expression analysis. Finally, the threedimensional (3D) structures of the HPs were constructed and further refined for comparative structure analysis and function relationship clarification. Twelve P. cryoconitis and four G. antarctica conserved HPs with significant thermal stress response functions that met crystallisation criteria were identified through in silica analysis. All target genes were successfully amplified and cloned in vitro. Three P. cryoconitis (PcbgSHPl, Pcbg5HP2, and Pcbg5HP12) recombinant proteins were overexpressed in the soluble forms at 16°C and subsequently purified using a twostep purification process. Three recombinant proteins from G. antarctica (GaHP2, GaHP3, and GaHP4) were overexpressed in soluble forms at 20°C, but only GaHP2 and GaHP3 were successfully purified. The ATPase assay showed protein activity at 4°C and 25°C for PcbgSHPl, Pcbg5HP2, Pcbg5HP12, GaHP2, and GaHP3, which thus clarified that protein activity is maintained at low and moderate temperatures. Meanwhile, lower citrate synthase aggregation at 43°C in the presence of either PcbgSHPl or GaHP2 suggested the characteristics of chaperone-like activity. The qPCR analysis revealed that these genes were expressed constitutively when cells were exposed to temperatures below or above their optimal growth temperature, indicating their involvement in cellular processes associated with thermal stress. Initial crystal formation was observed when purified Pcbg5HP2 proteins were incubated at 4°C, and reagent optimization revealed the formation of a plateshaped crystal in reagent 0.2 M potassium sodium tartrate tetrahydrate, 30% PEG/Ion, pH 7.4. This clarified the crystallisation potential of the HPs as predicted by bioinformatics analysis. Furthermore, comparative structural analysis demonstrated that the HPs exhibited cold-adapted traits, most notably increased flexibility in their 3D structures compared to their mesophilic or thermophilic counterparts. Concurrently, the presence of a disulphide bridge and aromatic clusters was attributed to PcbgSHPl and GaHP2's unusual protein stability and chaperone activity. Thus, this demonstrated that the HPs examined in this study adopted strategies to maintain a balance between molecular stability and structural flexibility, which contributed to their flexibility and ability to retain protein activities in an extreme environment. Conclusively, this study has established the structurefunction relationships of the HPs produced by P. cryoconitis and G. antarctica and provided crucial experimental evidence indicating their importance in thermal stress response.

Item Type:	Thesis (Post-Doctoral)
Keyword:	Genome data , Pedobacter cryoconitis , Glaciozyma antarctica
Subjects:	Q Science > QP Physiology > QP1-(981) Physiology > QP501-801 Animal biochemistry
Department:	INSTITUTE > Biotechnology Research Institute (BRI)
Depositing User:	DG MASNIAH AHMAD -
Date Deposited:	21 Jun 2023 09:23
Last Modified:	21 Jun 2023 09:23
URI:	https://eprints.ums.edu.my/id/eprint/35645

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