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

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Title: Modification of the Prosegment in Understanding its Role in the Folding and Function of PMII
Author: Jaafar, Ahmad
Department: Department of Food Science
Program: Food Science
Advisor: Yada, Rickey
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.
URI: http://hdl.handle.net/10214/8369
Date: 2014-08


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