Formation of a native‐like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor

Abstract

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 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 PαPβ, 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.