Issue 35, 2024

A growth type pathway for improving the folding of BPTI

Abstract

The in vitro oxidative folding of the protein bovine pancreatic trypsin inhibitor (BPTI) with oxidized dithiothreitol or glutathione has served as a paradigm for protein folding but could take weeks at physiological pH because of the need to escape from kinetic traps via a rearrangement type pathway. The two major kinetic traps are called N′ and N* and contain two of the three native disulfide bonds, which occur between residues 5 and 55, 30 and 51, and 14 and 38. N′ is missing the disulfide bond between residues 5 and 55 while N* is missing the disulfide bond between residues 30 and 51. By determining rate constant for the reactions of the kinetic traps N* and N′ and their mixed disulfides with glutathione and glutathione disulfide, many for the first time, we demonstrate that growth type pathways are feasible and could even be more efficient than rearrangement type pathways. Thus, formally unproductive pathways became productive. Interestingly, under physiological redox conditions both rearrangement and growth type pathways are important highlighting the redundancy of oxidative protein folding. With the new set of rate constants, modeling indicated that in vitro oxidative protein folding of BPTI via a growth type pathway using an oxidation, reduction and oxidation cycle would significantly improve protein folding efficiency, albeit under non-physiological redox conditions. With these changing conditions 91 ± 2% of native BPTI was achieved in 12 h compared to 83% native protein in 24 h using our previous best conditions of 5 mM GSSG and 5 mM GSH. Therefore, changing redox conditions via an oxidation, reduction and oxidation cycle may become an additional methodology for enhancing in vitro protein folding in aqueous solution.

Graphical abstract: A growth type pathway for improving the folding of BPTI

Supplementary files

Article information

Article type
Paper
Submitted
16 May 2024
Accepted
06 Aug 2024
First published
19 Aug 2024

Org. Biomol. Chem., 2024,22, 7180-7186

A growth type pathway for improving the folding of BPTI

Y. Wang, S. Mandumula and W. J. Lees, Org. Biomol. Chem., 2024, 22, 7180 DOI: 10.1039/D4OB00802B

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