Denaturant-dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds.
The equilibrium and kinetic behavior of the guanidine hydrochloride (Gdn-HCl) induced unfolding/refolding of four bovine pancreatic trypsin inhibitor (BPTI) mutants was examined by using ultraviolet difference spectroscopy. In three of the mutants, we replaced the buried 30-51 disulfide bond with alanine at position 51 and valine (Val30/Ala51), alanine (Ala30/Ala51), or threonine (Thr30/Ala51) at position 30. For the fourth mutant, the solvent-exposed 14-38 disulfide was substituted by a pair of alanines (Ala14/Ala38). All mutants retained the 5-55 disulfide. Experiments were performed under oxidizing conditions; thus, both the unfolded and folded forms retained two native disulfide bonds. Equilibrium experiments demonstrated that all four mutants were destabilized relative to wild-type BPTI. However, the stability of the 30-51 mutants increased with the hydrophobicity of the residue substituted at position 30. Kinetic experiments showed that all four mutants contained two minor slow refolding phases with characteristics of proline isomerization. The specific behavior of the phases depended on the location of the disulfide bonds. The major unfolding/refolding phase for each of the 30-51 mutants was more than an order of magnitude slower than for Ala14/Ala38 or for BPTI in which the 14-38 disulfide bond was specifically reduced and blocked with iodoacetamide [Jullien, M., & Baldwin, R. L. (1981) J. Mol. Biol. 145, 265-280]. Since this effect is independent of the stability of the protein, it is consistent with a model in which the proper docking of the interior residues of the protein is the rate-limiting step in the folding of these mutants.
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
M R
MR
Hurle
C B
CB
Marks
P A
PA
Kosen
S
S
Anderson
I D
ID
Kuntz
D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation
D004220:Disulfides; D006146:Guanidines; D007611:Aprotinin; D019791:Guanidine