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An engineered leucine zipper a position mutant with an unusual three‐state unfolding pathway
Author(s) -
Zhu Hai,
Celinski Scott A.,
Scholtz J. Martin,
Hu James C.
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.30901
Subject(s) - leucine zipper , heptad repeat , zipper , coiled coil , bzip domain , chemistry , dimer , mutant , protein folding , protein structure , crystallography , stereochemistry , biochemistry , biophysics , peptide sequence , biology , organic chemistry , algorithm , computer science , gene
The leucine zipper is a dimeric coiled‐coil structural motif consisting of four to six heptad repeats, designated ( abcdefg ) n . In the GCN4 leucine zipper, a position 16 in the third heptad is occupied by an Asn residue whereas the other a positions are Val residues. Recently, we have constructed variants of the GCN4 leucine zipper in which the a position Val residues were replaced by Ile. The folding and unfolding of the wild‐type GCN4 leucine zipper and the Val to Ile variant both adhere to a simple two‐state mechanism. In this study, another variant of the GCN4 leucine zipper was constructed by moving the single Asn residue from a position 16 to a position 9. This switch causes the thermal unfolding of the GCN4 leucine zipper to become three state. The unfolding pathway of this variant was determined by thermal denaturation, limited proteinase K digestion, and sedimentation equilibrium analysis. Our data are consistent with a model in which the variant first unfolds from its N terminus and changes the oligomerization specificity from a native dimer to a partially unfolded intermediate containing a mixture of dimers and trimers and then completely unfolds to unstructured monomers.