Complete folding of bovine pancreatic trypsin inhibitor with only a single disulfide bond.
In the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) at neutral pH, only two one-disulfide intermediates accumulate to a significant extent, namely [5-55] and [30-51]. In this paper we describe a recombinant model of [5-55], designated [5-55]Ala, which was made by replacing the cysteine residues not involved in the disulfide bond with alanine. As judged by two-dimensional NMR, [5-55]Ala folds into essentially the same conformation as native BPTI. Moreover, like native BPTI, [5-55]Ala inhibits trypsin stoichiometrically. Thus, the disulfide-bonded intermediate [5-55] corresponds not to a partially folded protein folding intermediate but rather to an essentially completely folded protein. This conclusion provides an explanation for many of the thermodynamic and kinetic properties of [5-55] in the folding pathway of BPTI.
Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research, Department of Chemistry, Nine Cambridge Center, MA 02142.
J P
JP
Staley
P S
PS
Kim
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000737:chemistry / Q000648:ultrastructure*; D002942:Circular Dichroism; D004220:Disulfides; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D004220:Disulfides; D011994:Recombinant Proteins; D007611:Aprotinin