Characterisation of structural and functional changes of human heat shock protein 47 wild-type and his-to-ala mutants
Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that localises in the endoplasmic reticulum (ER). The HSP47 is essential for a proper formation of the collagen triple helix. The HSP47 binds to the completely-folded collagen molecule and accompanies it from the ER to the Golg...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2021
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| Subjects: | |
| Online Access: | http://eprints.utm.my/id/eprint/101924/1/SitiMasturahZakeriaMFS2021.pdf |
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| Summary: | Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that localises in the endoplasmic reticulum (ER). The HSP47 is essential for a proper formation of the collagen triple helix. The HSP47 binds to the completely-folded collagen molecule and accompanies it from the ER to the Golgi apparatus (GA), where dissociation occurs and HSP47 is recycled back to the ER. The binding and release behaviour are triggered by the lower pH in the GA or the ER-Golgi intermediate compartment (ERGIC). Histidine has been suggested to be the trigger residue due to the side chain pKa value of around 6.1, similar to the HSP47-collagen dissociation pH. In this study, Histidine-to-alanine (HA) mutants of human HSP47 were constructed to elucidate the specific mechanism that governs HSP47 release from collagen at the molecular level, known as the “pH-switch mechanism”. The binding of three mutants were found to be affected, namely H220A, H335A and H368A. This study investigated the effects of these mutations to the human HSP47 binding strength, using collagen type I extracted from rat tail tendon and modified enzyme-linked immunosorbent assay (ELISA)-based binding assay. The secondary structure of mutant proteins was investigated using circular dichroism (CD) spectroscopy. The result showed that mutants were found to retain their binding ability to collagen, except for H220A. The H335A and H368A were found to have slightly lower binding affinity to collagen relative to the wild-type (WT) (Kd = 50.76 nM), with a dissociation constant (Kd ) of 152.9 nM and 131.3 nM respectively. In addition to the lost in collagen binding, H220A was also significantly more thermostable compared to the other mutants, that have similar thermostability to WT. The secondary structure of the HA mutants at alkaline pH was found to differ slightly from WT, with H368A showed a perturbed pH-induced secondary structural changes. In conclusion, the binding strength of the human HSP47 HA mutants was successfully elucidated and the structural changes caused by the mutations were described. The H220 was suggested to be important for binding with collagen while H368 was important for the release mechanism. |
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