Relationship between Folding and Function in a Sequence-Specific Miniature DNA-Binding Protein

Abstract

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, wellfolded protein avian pancreatic polypeptide acts as a scaffold to present and stabilize an R-helical or PPII-helical recognition epitope. The first miniature protein designed in this way, a molecule called p007, presents the R-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 R-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 R-helicity and affinity, we observe a limited correlation between R-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 R-helical structure, is the primary determinant of p007 function.The development of general strategies for the selective recognition of macromolecular targets remains a major challenge for chemical biology and a fundamental postgenome goal. Molecules capable of tight and selective macromolecular recognition have utility in the validation of potential therapeutic targets and can, in certain cases, function as therapeutics in their own right (1-3). Our laboratory has developed a general approach toward the design of small, well-folded proteins that bind macromolecular targets with high affinity and selectivity (4-11). This approach is often referred to as protein grafting, and the molecules that result are called miniature proteins: miniature because they contain fewer than 40 amino acids and proteins because they often fold cooperatively. In a protein grafting experiment, those residues that comprise a natural R-helical or type II polyproline (PPII)-helical recognition epitope are introduced onto the solvent-exposed R-or PPII-helical face of the small yet stable protein avian pancreatic polypeptide (aPP) 1 (12, 13). Our laboratory has used this procedure, often in combination with directed evolution, to engineer miniature protein ligands with high affinity for a variety of targets including the duplex DNAs recognized by GCN4 and CREB (4, 5) and the Q50K engrailed homeodomain (7), the antiapoptotic proteins Bcl-X L (6) and Bcl-2 (10), the oncoprotein hDM2 (manuscript in prepara...

Direct force measurement of single DNA–peptide interactions using atomic force microscopy

“…In our DNA–peptide complex system, the rupture force versus the logarithm of the loading rate was fitted with a single linear plot, suggesting that there is a single barrier between bound and unbound states. The calculated free energy based upon K d (1.6 nM) (Yang and Schepartz ) was 20.3 k B T , and the estimated energy barrier using the Arrhenius equation was 21.9 k B T . Our values of k off and ∆ x obtained from a linear fit were 0.32/s and 0.74 nm, respectively.…”

“…This DNA–peptide complex has the equilibrium dissociation constant K d = 1.6 nM (Chin and Schepartz ), which is in the range of the values of the antigen–antibody complex (0.01~100 nM) (Maynard and Georgiou, ). The K d of p007 that selectively binds to DNA with the ATGAC sequence was 200~800 times lower than that of p007 that binds to analogous DNA with two‐base‐pair mutations in ATGAC and 4000‐fold lower than that of p007 that nonspecifically binds to calf thymus DNA (Chin and Schepartz, ; Yang and Schepartz, ). This sequence‐specific strong interaction between a small peptide and DNA can be utilized to create mechanically stable well‐defined DNA–peptide hybrid nanostructures in addition to therapeutic applications.…”

“…For example, regulation of cellular processes requires the specific interactions among proteins that are stimulated or inhibited by posttranslational modifications or effectors (Pawson and Nash, 2003). The specific recognitions with very high affinity are widely utilized in biotechnology in the areas of purification, diagnostics (Yu et al, 2006;Ozkumur et al, 2008), and drug screening (Huang, 2000;Yang and Schepartz, 2005). Interactions between DNA and protein are one of the fundamental reactions for many intracellular processes (Verdine and Norman, 2003).…”

Nanofabrication for Molecular Scale Devices

“…It should be pointed out that the DNA-protein interaction may be of general importance in fabricating molecular electronic devices (not just applications like biosensors as might be expected) because of the patterning function briefly illustrated here. Binding and interaction between DNA and proteins are being more and more elucidated by ongoing research (Dixit et al, 2005;Yang and Schepartz, 2005;Hu and Shklovskii, 2006;Sun et al, 2006).…”

Miniature Protein Inhibitors of the p53–hDM2 Interaction

“…[1,27] In an attempt to introduce additional contacts within hDM2's p53AD-binding pocket, library 3 was constructed to include 1.1 variants with diversity at four positions C-terminal to the hDM2 recognition motif. Each library contained 2 10 8 unique transformants, which is sufficient for evaluating DNA-sequence space with 100 % confidence.…”