Formation of a native-like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor.
In the folding of bovine pancreatic trypsin inhibitor (BPTI), the single-disulfide intermediate [30-51] plays a key role. We have investigated a recombinant analog of [30-51] using a 2-dimensional nuclear magnetic resonance (2D-NMR). This recombinant analog, named [30-51]Ala, contains a disulfide bond between Cys-30 and Cys-51, but contains alanine in place of the other cysteines in BPTI to prevent the formation of other intermediates. By 2D-NMR, [30-51]Ala consists of 2 regions-one folded and one predominantly unfolded. The folded region resembles a previously characterized peptide model of [30-51], named P alpha P beta, that contains a native-like subdomain with tertiary packing. The unfolded region includes the first 14 N-terminal residues of [30-51] and is as unfolded as an isolated peptide containing these residues. Using protein dissection, we demonstrate that the folded and unfolded regions of [30-51]Ala are structurally independent. The partially folded structure of [30-51]Ala explains many of the properties of authentic [30-51] in the folding pathway of BPTI. Moreover, direct structural characterization of [30-51]Ala has revealed that a crucial step in the folding pathway of BPTI coincides with the formation of a native-like subdomain, supporting models for protein folding that emphasize the formation of cooperatively folded subdomains.
Staley
J P
JP
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02142.
Kim
P S
PS
D000409:Alanine / Q000737:chemistry; D000818:Animals; D007611:Aprotinin / Q000737:chemistry*; D002417:Cattle; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry
D004220:Disulfides; D011994:Recombinant Proteins; D007611:Aprotinin; D003545:Cysteine; D000409:Alanine