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Shonhai, A., Zininga, T. Dongola, Tendamudzimu Harmfree 2021 2022-09-13T14:25:32Z 2022-09-13T14:25:32Z 2022-07-15 Dongola, T. H. (2021) In cellulo and biophysical exploration of the role of GGMP residues of Hsp70. University of Venda. South Africa.<http://hdl.handle.net/11602/2261>. http://hdl.handle.net/11602/2261 Dongola TH. In cellulo and biophysical exploration of the role of GGMP residues of Hsp70. []. , 2022 [cited yyyy month dd]. Available from: http://hdl.handle.net/11602/2261 Dongola, T. H. (2022). <i>In cellulo and biophysical exploration of the role of GGMP residues of Hsp70</i>. (). . Retrieved from http://hdl.handle.net/11602/2261 Dongola, Tendamudzimu Harmfree. <i>"In cellulo and biophysical exploration of the role of GGMP residues of Hsp70."</i> ., , 2022. http://hdl.handle.net/11602/2261 TY - Dissertation AU - Dongola, Tendamudzimu Harmfree AB - Hsp70 is a prominent molecular chaperone. Structurally, Hsp70 is composed of two domains, C-terminus substrate binding domain (SBD) and nucleotide binding domain (NBD). The NBD of Hsp70 is responsible for its ATPase activity. Some Hsp70s of parasites particularly those of apicomplexa are marked by GGMP residues. For example, Plasmodium falciparum Hsp70-1 (PfHsp70-1) which occurs in the cytosol and nucleus harbors seven GGMP repeats on the C terminus upstream of the EEVD motif. The function of GGMP residues of Hsp70 is largely unclear but they were recently reported to be involved in substrate and co-chaperone binding. Therefore, the main aim of this study was to investigate the role of the GGMP residues of Hsp70s using E. coli Hsp70 (DnaK) and chimeric protein, KPf as models. Chimeric protein KPf is made up of NBD of DnaK and SBD of PfHsp70-1. E. coli DnaK lacks the GGMP residues that are present in PfHsp70-1. DnaK-G (DnaK modified to include GGMP residues) was created to elucidate the function of these residues. Exogenously expressed KPf and DnaK are known to reverse the thermosensitivity of E. coli dnaK756 cells. The native DnaK of E. coli dnaK756 cells is functionally compromised making this strain heat sensitive. KPf617-647 and KPfΔG mutants were previously created by conservative substitution and deleting the GGMP residues of KPf, respectively. Circular dichroism and tryptophan fluorescence highlighted that the insertion of GGMP residues did not drastically change the secondary and tertiary structure of DnaK. Cytoprotection of E. coli dnaK756 cells could not be recovered when cells were heterologous expressing DnaK-G. DnaK recovered the cytoprotection of the same cells as expected. Furthermore, mutations on GGMP residues adversely impacted the chaperone function of KPf with respect to cytoprotection of E. coli dnaK756 cells. On the other hand, the absence of the GGMP residues results in KPf losing its chaperone function. This suggests that the SBD of PfHsp70-1 requires the GGMP residues to function, whereas E. coli DnaK does not require these residues. It was further noted that insertion of the GGMP residues into DnaK led to toxicity in E. coli dnaK103 cells under permissive growth temperature. A proteomic study conducted to establish the functional deficiencies of DnaK-G established that the presence of the GGMP residues compromised the capability of DnaK-G to bind some substrates. Altogether the findings suggest that GGMP repeats account for the specialized function of PfHsp70-1 in distinction to that of E. coli DnaK. DA - 2022-07-15 DB - ResearchSpace DP - Univen KW - Heat shock proteins KW - Molecular chaperones KW - GGMP residues Dnak KW - Complementation assay LK - https://univendspace.univen.ac.za PY - 2022 T1 - In cellulo and biophysical exploration of the role of GGMP residues of Hsp70 TI - In cellulo and biophysical exploration of the role of GGMP residues of Hsp70 UR - http://hdl.handle.net/11602/2261 ER - MSc (Biochemistry) Department of Biochemistry and Microbiology Hsp70 is a prominent molecular chaperone. Structurally, Hsp70 is composed of two domains, C-terminus substrate binding domain (SBD) and nucleotide binding domain (NBD). The NBD of Hsp70 is responsible for its ATPase activity. Some Hsp70s of parasites particularly those of apicomplexa are marked by GGMP residues. For example, Plasmodium falciparum Hsp70-1 (PfHsp70-1) which occurs in the cytosol and nucleus harbors seven GGMP repeats on the C terminus upstream of the EEVD motif. The function of GGMP residues of Hsp70 is largely unclear but they were recently reported to be involved in substrate and co-chaperone binding. Therefore, the main aim of this study was to investigate the role of the GGMP residues of Hsp70s using E. coli Hsp70 (DnaK) and chimeric protein, KPf as models. Chimeric protein KPf is made up of NBD of DnaK and SBD of PfHsp70-1. E. coli DnaK lacks the GGMP residues that are present in PfHsp70-1. DnaK-G (DnaK modified to include GGMP residues) was created to elucidate the function of these residues. Exogenously expressed KPf and DnaK are known to reverse the thermosensitivity of E. coli dnaK756 cells. The native DnaK of E. coli dnaK756 cells is functionally compromised making this strain heat sensitive. KPf617-647 and KPfΔG mutants were previously created by conservative substitution and deleting the GGMP residues of KPf, respectively. Circular dichroism and tryptophan fluorescence highlighted that the insertion of GGMP residues did not drastically change the secondary and tertiary structure of DnaK. Cytoprotection of E. coli dnaK756 cells could not be recovered when cells were heterologous expressing DnaK-G. DnaK recovered the cytoprotection of the same cells as expected. Furthermore, mutations on GGMP residues adversely impacted the chaperone function of KPf with respect to cytoprotection of E. coli dnaK756 cells. On the other hand, the absence of the GGMP residues results in KPf losing its chaperone function. This suggests that the SBD of PfHsp70-1 requires the GGMP residues to function, whereas E. coli DnaK does not require these residues. It was further noted that insertion of the GGMP residues into DnaK led to toxicity in E. coli dnaK103 cells under permissive growth temperature. A proteomic study conducted to establish the functional deficiencies of DnaK-G established that the presence of the GGMP residues compromised the capability of DnaK-G to bind some substrates. Altogether the findings suggest that GGMP repeats account for the specialized function of PfHsp70-1 in distinction to that of E. coli DnaK. NRF 1 online resource (72 leaves) : color illustrations en University of Venda Heat shock proteins UCTD Molecular chaperones GGMP residues Dnak Complementation assay 572.6 Molecular chaperones Protein Heat shock proteins In cellulo and biophysical exploration of the role of GGMP residues of Hsp70 Dissertation open.access