An engineered leucine zipper a position mutant with an unusual three-state unfolding pathway.
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.
Zhu
H
H
Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843, USA.
Celinski
S A
SA
Scholtz
J M
JM
Hu
J C
JC
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014508:Urea / Q000737:chemistry
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014508:Urea; D011494:Protein Kinases