Folding of Bovine Pancreatic Trypsin Inhibitor (BPTI) is Faster using Aromatic Thiols and their Corresponding Disulfides

Authors

Ram Prasad Marahatta, Florida International UniversityFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Chemistry

First Advisor's Name

Watson J. Lees

First Advisor's Committee Title

Committee chair

Second Advisor's Name

Kevin O'Shea

Second Advisor's Committee Title

Committee member

Third Advisor's Name

Prem P. Chapagain

Third Advisor's Committee Title

Committee member

Fourth Advisor's Name

Francisco Alberto Fernandez Lima

Fourth Advisor's Committee Title

Committee member

Fifth Advisor's Name

Xiaotang Wang

Fifth Advisor's Committee Title

Committee member

Keywords

Protein folding, glutathione (GSH), glutathione disulfide (GSSG), aromatic thiols, aromatic disulfides, BPTI, HPLC, molecular dynamics (MD), Targeted MD (TMD), conformational folding

Date of Defense

11-17-2017

Abstract

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*, N^SH were formed and native protein was ultimately obtained. We concluded that there could be multiple pathways of conformational folding which influence oxidative folding.

Identifier

FIDC004036

Recommended Citation

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.
https://digitalcommons.fiu.edu/etd/3530