Protein Science, Vol 3, Issue 10 1822-1832, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

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

J. P. STALEY and P. S. KIM
Department of Chemistry Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142 Present address: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California 94143-0448.

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 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.
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