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

• Published in: Protein science Vol. 10; no. 1; pp. 24 - 33
• Main Authors: Zhu, Hai, Celinski, Scott A., Scholtz, J. Martin, Hu, James C.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.01.2001
• Subjects: Amino Acid Sequence; DNA-Binding Proteins; folding intermediate; Fungal Proteins - chemistry; Fungal Proteins - genetics; Hot Temperature; Leucine Zippers; Molecular Sequence Data; Mutation; Protein Denaturation; Protein Engineering; Protein Folding; Protein Kinases - chemistry; Protein Kinases - genetics; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; three‐state unfolding; Urea - chemistry
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.30901

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.