Relationship between folding and function in a sequence-specific miniature DNA-binding protein.
Previously, we have described a miniature protein-based approach to the design of molecules that bind DNA or protein surfaces with high affinity and specificity. In this approach, the small, well-folded protein avian pancreatic polypeptide acts as a scaffold to present and stabilize an alpha-helical or PPII-helical recognition epitope. The first miniature protein designed in this way, a molecule called p007, presents the alpha-helical recognition epitope found on the bZIP protein GCN4 and binds DNA with nanomolar affinity and exceptional specificity. In this work we use alanine-scanning mutagenesis to explore the contributions of 29 p007 residues to DNA affinity, specificity, and secondary structure. Virtually every residue within the p007 alpha-helix, and most residues within the p007 PPII helix, contribute to both DNA affinity and specificity. These residues include those introduced to make specific and nonspecific DNA contacts, as well as those that complete the miniature protein core. Moreover, there exists a direct correlation between the affinity of a p007 variant for specific DNA and the ability of that variant to select for specific DNA over nonspecific DNA. Although we observe no correlation between alpha-helicity and affinity, we observe a limited correlation between alpha-helicity and sequence specificity that emphasizes the role of coupled binding/folding in the function of p007. Our results imply that formation of a highly evolved set of protein.DNA contacts in the context of a well-packed hydrophobic core, and not the extent of intrinsic alpha-helical structure, is the primary determinant of p007 function.
Yang
Loretta
L
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Schepartz
Alanna
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D050976:Basic-Leucine Zipper Transcription Factors; D001667:Binding, Competitive; D002942:Circular Dichroism; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D019020:Directed Molecular Evolution; D051790:G-Box Binding Factors; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry; D017433:Protein Structure, Secondary; D020218:Response Elements; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry
D050976:Basic-Leucine Zipper Transcription Factors; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases