Consolidating Critical Binding Determinants by Noncyclic Rearrangement of Protein Secondary Structure

• Published in: Proceedings of the National Academy of Sciences - PNAS 2005-02, Vol.102 (7), p.2305-2309
• Main Authors: Tabtiang, Ramon K., Cezairliyan, Brent O., Grant, Robert A., Cochrane, Jesse C., Sauer, Robert T.
• Format: Article
• Language: English
• Subjects: Bacteriophage P22 - chemistry; Bacteriophage P22 - genetics; Biochemistry; Biological Sciences; Crystal structure; Crystallography, X-Ray; Crystals; Datasets; Deoxyribonucleic acid; Dimers; DNA; DNA, Viral - genetics; DNA, Viral - metabolism; Kinetics; Models, Molecular; Molecules; Protein Binding; Protein Folding; Protein refolding; Protein Structure, Secondary; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Repressor Proteins - chemistry; Repressor Proteins - genetics; Repressor Proteins - metabolism; Structural members; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Viral Regulatory and Accessory Proteins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0409562102

We designed a single-chain variant of the Arc repressor homodimer in which the β strands that contact operator DNA are connected by a hairpin turn and the α helices that form the tetrahelical scaffold of the dimer are attached by a short linker. The designed protein represents a noncyclic permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc), in which the two subunits are fused by a single linker. The permuted protein binds operator DNA with nanomolar affinity, refolds on the submillisecond time scale, and is as stable as Arc-L1-Arc. The crystal structure of the permuted protein reveals an essentially wild-type fold, demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure. Noncyclic rearrangement of secondary structure may allow grouping of critical activesite residues in other proteins and could be a useful tool for protein design and minimization.