ABSTRACT
We describe a novel approach to the design of a metal-triggered conformational switch. Specifically, two distinct protein-folding motifs were merged into one polypeptide sequence. The target structures were an alpha-helical coiled-coil trimer and zinc-bound monomer. Solution-phase spectroscopic, sedimentation, and binding studies confirmed the key aspects of the design. Both forms of the peptide were cooperatively folded, and the switch between them was reversible. This design process potentially presents a novel route to peptide-based biosensors.
Subject(s)
Chlorides/chemistry , Peptides/chemistry , Protein Folding , Zinc Compounds/chemistry , Zinc/chemistry , Binding Sites , Chlorides/metabolism , Circular Dichroism , Edetic Acid/chemistry , Edetic Acid/metabolism , Peptides/metabolism , Protein Structure, Secondary , Spectroscopy, Fourier Transform Infrared , Temperature , Zinc/metabolism , Zinc Compounds/metabolismABSTRACT
Zinc binding motifs have received much attention in the area of protein design. Here, we have tested the suitability of a recently discovered nonnative zinc binding structure as a protein design scaffold. A series of multiple alanine mutants was created to investigate the minimal requirements for folding, and solution structures of these mutants showed that the original fold was maintained, despite changes in approximately 50% of the sequence. We next attempted to transplant binding faces from chosen bimolecular interactions onto one of these mutants, and many of the resulting "chimeras" were shown to adopt a native-like fold. These results both highlight the robust nature of small zinc binding domains and underscore the complexity of designing functional proteins, even using such small, highly ordered scaffolds as templates.
Subject(s)
Alanine/genetics , Carrier Proteins/chemistry , Mutagenesis , Zinc/chemistry , Amino Acid Sequence , Molecular Sequence Data , Molecular Structure , Mutation/genetics , Peptides/chemistry , Peptides/genetics , Protein Conformation , Protein Folding , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Solutions/chemistryABSTRACT
Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.
