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Modification of the Prosegment in Understanding its Role in the Folding and Function of PMII

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dc.contributor.advisor Yada, Rickey
dc.contributor.author Jaafar, Ahmad
dc.date.accessioned 2014-09-02T15:11:30Z
dc.date.available 2015-02-25T06:00:16Z
dc.date.copyright 2014-08
dc.date.created 2014-08-26
dc.date.issued 2014-09-02
dc.identifier.uri http://hdl.handle.net/10214/8369
dc.description.abstract This thesis explores the folding and activation of plasmepsin II (PMII) and the role of its prosegment (PS) on structure – function. Three different constructs of PMII were designed with PSs of varying lengths: extended PSPMII with a 60 residue PS, truncated PSPMII with a 48 residue PS, and NoProPMII with no PS. Extended and truncated PSPMII produced mature enzyme with similar conformation. NoProPMII mature, however, showed improper folding as indicated by low thermal stability, a more solvent-exposed conformation, an 11-fold reduction in the activity assay, and a lower pepstatin-A requirement for complete inhibition. In addition, the PS length was discovered to affect the activation of PMII. Extended PSPMII produced mature enzyme with an extra two PS residues (+2 PMII mature) whereas truncated PSPMII produced mature enzyme with an extra 12 PS residues (+12 PMII mature). The role of a PS in PMII folding was further investigated by conducting folding kinetic studies on PMII. It was found that the native PMII (Np) did not fold at the lowest free energy, but was kinetically stabilized. Upon unfolding, Np formed a thermodynamically stable, yet inactive refolded state (Rp). Np was characterized to have a slow rate of unfolding and folding, as indicated by large free energy barriers to unfold and fold of 24.50 and 25.12 kcal/mol, respectively. In the presence of the extended PS (60 residues), the energy landscape was shifted and the activation energy barrier was lowered to 12.37 kcal/mol, which enhanced the folding rate by approximately 18,550 times. To elucidate the effect of PS residue length on PMII folding, structural analysis and in silico simulation were conducted on the two zymogens of PMII: extended PSPMII and truncated PSPMII. Both zymogens appeared to be in a more solvent-exposed conformation as compared to their mature forms. Due to the oppositely charged residues between the main protein body and the PS, it appeared that the interactions between both structures were driven by electrostatic forces with 27 and 34 interactions (H-bonds and salt-bridges) measured between the main PMII body and truncated and extended PS, respectively. en_US
dc.description.sponsorship Malaysian Ministry of Education (MOE) and Universiti Putra Malaysia (UPM) en_US
dc.language.iso en en_US
dc.subject Plasmepsin II en_US
dc.subject prosegment en_US
dc.subject zymogen en_US
dc.subject kinetic stability en_US
dc.subject catalyzed folding en_US
dc.title Modification of the Prosegment in Understanding its Role in the Folding and Function of PMII en_US
dc.type Thesis en_US
dc.degree.programme Food Science en_US
dc.degree.name Doctor of Philosophy en_US
dc.degree.department Department of Food Science en_US
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