Folding of Bovine Pancreatic Trypsin Inhibitor (BPTI) is Faster using Aromatic Thiols and their Corresponding Disulfides
Doctor of Philosophy (PhD)
First Advisor's Name
Watson J. Lees
First Advisor's Committee Title
Second Advisor's Name
Second Advisor's Committee Title
Third Advisor's Name
Prem P. Chapagain
Third Advisor's Committee Title
Fourth Advisor's Name
Francisco Alberto Fernandez Lima
Fourth Advisor's Committee Title
Fifth Advisor's Name
Fifth Advisor's Committee Title
Protein folding, glutathione (GSH), glutathione disulfide (GSSG), aromatic thiols, aromatic disulfides, BPTI, HPLC, molecular dynamics (MD), Targeted MD (TMD), conformational folding
Date of Defense
Improvement in the in vitro oxidative folding of disulfide-containing proteins, such as extracellular and pharmaceutically important proteins, is required. Traditional folding methods using small molecule aliphatic thiol and disulfide, such as glutathione (GSH) and glutathione disulfide (GSSG) are slow and low yielding. Small molecule aromatic thiols and disulfides show great potentiality because aromatic thiols have low pKa values, close to the thiol pKa of protein disulfide isomerase (PDI), higher nucleophilicity and good leaving group ability. Our studies showed that thiols with a positively charged group, quaternary ammonium salts (QAS), are better than thiols with negatively charged groups such as phosphonic acid and sulfonic acid for the folding of bovine pancreatic trypsin inhibitor (BPTI). An enhanced folding rate of BPTI was observed when the protein was folded with a redox buffer composed of a QAS thiol and its corresponding disulfide.
Quaternary ammonium salt (QAS) thiols and their corresponding disulfides with longer alkyl side chains were synthesized. These QAS thiols and their corresponding disulfides are promising small molecule thiols and disulfides to fold reduced BPTI efficiently because these thiols are more hydrophobic and can enter the core of the protein.
Conformational changes of disulfide-containing proteins during oxidative folding influence the folding pathway greatly. We performed the folding of BPTI using targeted molecular dynamics (TMD) simulation and investigated conformational changes along with the folding pathway. Applying a bias force to all atoms versus to only alpha carbons and the sulfur of cysteines showed different folding pathways. The formation of kinetic traps N' and N* was not observed during our simulation applying a bias force to all atoms of the starting structure. The final native conformation was obtained once the correct antiparallel β-sheets and subsequent Cys14-Cys38 distance were decreased to a bond distance level. When bias force was applied to only alpha carbons and the sulfur of cysteines, the distance between Cys14-Cys38 increased and decreased multiple times, a structure similar to the confirmation of N*, NSH were formed and native protein was ultimately obtained. We concluded that there could be multiple pathways of conformational folding which influence oxidative folding.
Marahatta, Ram Prasad, "Folding of Bovine Pancreatic Trypsin Inhibitor (BPTI) is Faster using Aromatic Thiols and their Corresponding Disulfides" (2017). FIU Electronic Theses and Dissertations. 3530.
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