Disulfide bond-coupled folding of bovine pancreatic trypsin inhibitor derivatives missing one or two disulfide bonds.
The disulfide bond-coupled folding and unfolding mechanism (at pH 8.7, 25 degrees C in the presence of oxidized and reduced dithiothreitol) was determined for a bovine pancreatic trypsin inhibitor mutant in which cysteines 30 and 51 were replaced with alanines so that only two disulfides, between cysteines 14 and 38 and cysteines 5 and 55, remain. Similar studies were made on a chemically-modified derivative of the mutant retaining only the 5-55 disulfide. The preferred unfolding mechanism for the Ala30/Ala51 mutant begins with reduction of the 14-38 disulfide. An intramolecular rearrangement via thiol-disulfide exchange, involving the 5-55 disulfide and cysteines 14 and/or 38, then occurs. At least five of six possible one-disulfide bond species accumulate during unfolding. Finally, the disulfide of one or more of the one-disulfide bond intermediates (excluding that with the 5-55 disulfide) is reduced giving unfolded protein. The folding mechanism seems to be the reverse of the unfolding mechanism; the observed folding and unfolding reactions are consistent with a single kinetic scheme. The rate constant for the rate-limiting intramolecular folding step--rearrangements of other one-disulfide bond species to the 5-55 disulfide intermediate--seems to depend primarily on the number of amino acids separating cysteines 5 and 55 in the unfolded chain. The energetics and kinetics of the mutant's folding mechanism are compared to those of wild-type protein [Creighton, T. E., & Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497] and a mutant missing the 14-38 disulfide [Goldenberg, D. P. (1988) Biochemistry 27, 2481]. The most striking effects are destabilization of the native structure and a large increase in the rate of unfolding.
Kosen
P A
PA
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Marks
C B
CB
Falick
A M
AM
Anderson
S
S
Kuntz
I D
ID
D000409:Alanine / Q000737:chemistry; D000478:Alkylation; D007611:Aprotinin / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D011487:Protein Conformation
D004220:Disulfides; D007611:Aprotinin; D005978:Glutathione; D003545:Cysteine; D000409:Alanine; D004229:Dithiothreitol