UNFOLDED BPTI VARIANTS WITH A SINGLE DISULFIDE BOND HAVE DIMINISHED NONNATIVE STRUCTURE DISTANT FROM THE CROSS-LINK

Background: NMR studies of denatured states, both fully unfolded and p artially folded, give insight into the conformations and interactions favored in initial stages of folding, and in early intermediates forme d during folding, We have characterized non-random structures favored in unfolded, reduced BPTI[1], and in partially folded BPTI[2], Here, w e report NMR-detected structure of two analogs of unfolded BPTI with o ne native 14-38 disulfide bond, Results: Analogs Y21A[14-38](Abu) and Y23A[14-38](Abu), obtained by chemical synthesis of [14-38](Abu) With Y21 or Y23 replaced by alanine, are models for unfolded BPTI with 14-3 8 the only disulfide, Compared to unfolded BPTI with all three disulfi des broken, the unfolded 14-38 BPTI analogs have numerous differences, including loss of non-native, turn-like conformations for beta 2 resi dues, diminished non-native aromatic-aliphatic NOEs, and increased int ermediate chemical exchange of residues that have native-like conforma tions in partially folded BPTI, Although the Y21A and Y23A analogs hav e similar CD and NMR properties, specific differences in NOE patterns and in exchange broadening are observed. Conclusions: Changes in unfol ded BPTI associated with formation of the 14-38 disulfide bond are con sistent with less non-native structure, and more native-like structure , in residues composing the stable core of antiparallel beta-sheet in partially folded BPTI, Specific differences between Y21A[14-38](Abu) a nd Y23A[14-38](Abu) indicate that replacement of Y23 results in less o rdered structure than replacement of Y21. (C) Current Biology Ltd