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An engineered leucine zipper a position mutant with an unusual three-state unfolding pathway

¹ Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843, USA
² Department of Medical Biochemistry and Genetics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843, USA

Reprint requests to: James C. Hu, Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, TX 77843-2128, USA; e-mail: jimhu{at}tamu.edu; fax: 979-845-9274.

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.

Keywords: Leucine zippers; protein folding; folding intermediate; three-state unfolding

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