Methodology for optimizing functional miniature proteins based on avian pancreatic polypeptide using phage display

Chin, Jason W.; Grotzfeld, Robert M.; Fabian, Miles A.; Schepartz, Alanna

doi:10.1016/s0960-894x(01)00139-1

Cited by 34 publications

(26 citation statements)

References 19 publications

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“…Protein grafting has proven to be a versatile solution to the problem of macromolecular recognition, having been used previously to identify miniature protein ligands for both the DNA major groove [12][13][14]16 and deep protein clefts. 15 The complex formed between the α-helical phosphorylated KID domain of CREB and the KIX domain of CBP 30 provided us with a unique context in which to address two questions about the generality and utility of protein grafting in the design of ligands for protein surfaces.…”

Section: Discussionmentioning

confidence: 99%

“…10,11 Thus, small proteins with well-defined three-dimensional structures and finely tuned functional properties are perhaps ideally suited for protein surface recognition and disruption of protein•protein interfaces. Our laboratory has recently described a general solution, called protein grafting, for the identification of highly functional miniature proteins by stabilization of α-helical binding epitopes on a protein scaffold [12][13][14][15][16] ( Figure 1A). In protein grafting, those residues that comprise a functional epitope are grafted onto the solvent-exposed α-helical face of the small yet stable protein avian pancreatic polypeptide (aPP).…”

Section: Introductionmentioning

confidence: 99%

“…17 This procedure, often in combination with molecular evolution, identifies miniature protein ligands with high affinity and specificity for macromolecular targets. Initially developed in the context of DNA recognition, [12][13][14] protein grafting has been applied recently to the identification of miniature proteins with nanomolar affinities for the related anti-apoptotic proteins Bcl-2 and Bcl-X L . 5,15 Although interesting, the application of protein grafting in this case represented a rather conservative step, as Bcl-2 and Bcl-X L each contain a deep (approximately 7 Å at deepest point) hydrophobic groove which recognizes α-helical regions of protein partners such as Bak.…”

Section: Introductionmentioning

confidence: 99%

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Molecular Recognition of Protein Surfaces: High Affinity Ligands for the CBP KIX Domain

2003

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Potent and specific inhibitors of protein•protein interactions have significant potential both as therapeutic compounds and biological tools, yet discovery of such molecules remains a significant challenge. Whereas small molecules typically bind proteins in small, well-defined, deep clefts, proteins generally recognize each other through formation of large, heterogeneous complementary surfaces. Our laboratory has recently described a general solution, called protein grafting, for the identification of highly functional miniature proteins by stabilization of α-helical binding epitopes on a protein scaffold. In protein grafting, those residues that comprise a functional epitope are grafted onto the solvent-exposed α-helical face of the small yet stable protein avian pancreatic polypeptide (aPP). In this study, we use protein grafting in combination with molecular evolution by phage display to identify phosphorylated peptide ligands that recognize the shallow surface of CBP KIX with high nanomolar to low micromolar affinity. Furthermore, we show that grafting of the CBP KIX-binding epitope of CREB KID onto the aPP scaffold yields molecules capable of high affinity recognition of CBP KIX even in the absence of phosphorylation. Importantly, both classes of designed ligands exhibit high specificity for the target CBP KIX domain over carbonic anhydrase and calmodulin, two unrelated proteins that bind hydrophobic or α-helical molecules that might be encountered in vivo.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular Recognition of Protein Surfaces: High Affinity Ligands for the CBP KIX Domain

2003

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“…However, PPBR4 failed to bind specific DNA detectably at room temperature, and circular dichroism (CD) and NMR experiments indicated that it possessed only nascent R-helicity in the absence of DNA (19). Building on the hypothesis that the lack of structure in PPBR4 resulted from disruption of the hydrophobic core, in the second stage of design (20) we used phage display to combinatorially vary residues on the N-terminal PPII helix that could repack the core. This selection led to a significantly more helical (21) miniature protein, p007, which binds specific DNA with picomolar affinity at 4°C and with nanomolar affinity at 25°C and which displays greatly enhanced specificity when compared with PPBR4 ( Figure 1A) (5).…”

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confidence: 99%

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

Yang

Schepartz

2005

Biochemistry

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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...

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“…They dissect the residues required for sequence-specific DNA binding and graft them onto the well-folding, but nonfunctional, aPP α-helix. A library of proteins is thus generated by solid-phase peptide synthesis or phage display [18], and functional selection follows. Using their protein-grafting strategy, the Schepartz group has generated an aPP variant that mimics GCN4's high DNA-binding affinity and specificity [17], another variant that comprises the DNA contact residues from the engrailed homeodomain also capable of high DNA-binding affinity and specificity [19], and miniature proteins that bind to human proteins Bcl-2 and Bcl-X L [20].…”

Section: Introductionmentioning

confidence: 99%

Minimalist proteins: Design of new molecular recognition scaffolds

Shin

2004

Pure and Applied Chemistry

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We hypothesize that we can exploit what Nature has already evolved by manipulating the α-helix molecular recognition scaffold. Therefore, minimalist proteins capable of sequence-specific, high-affinity binding of DNA were generated to probe how proteins are used and can be used to recognize DNA. The already minimal basic region/leucine zipper motif (bZIP) of GCN4 was reduced to an even more simplified structure by substitution with alanine residues-hence, a generic, Ala-based, helical scaffold. The proteins generated, wt bZIP, 4A, 11A, and 18A, contain 0, 4, 11, and 18 alanine mutations in their DNA-binding basic regions, respectively. All alanine mutants still retain α-helical structure and DNA-binding function, despite loss of virtually all Coulombic protein-DNA interactions. Mass spectrometry allowed characterization of proteins and post-translational modifications. Fluorescence anisotropy and DNase I footprinting were used to measure in situ binding of these mutant proteins to DNA duplexes containing target sites AP-1 (5′-TGACTCA-3′), ATF/CREB (5′-TGACGTCA-3′), or nonspecific DNA. The roles of van der Waals and Coulombic interactions toward binding specificity and affinity are being investigated. Thus, both DNA-binding specificity and affinity are maintained in all our bZIP derivatives. This Ala-rich scaffold may be useful in design and synthesis of small, α-helical proteins with desired DNA-recognition properties.

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Methodology for optimizing functional miniature proteins based on avian pancreatic polypeptide using phage display

Cited by 34 publications

References 19 publications

Molecular Recognition of Protein Surfaces: High Affinity Ligands for the CBP KIX Domain

Molecular Recognition of Protein Surfaces: High Affinity Ligands for the CBP KIX Domain

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

Minimalist proteins: Design of new molecular recognition scaffolds

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