Highly Specific DNA Recognition by a Designed Miniature Protein

Zondlo, Neal J.; Schepartz, Alanna

doi:10.1021/ja990968z

Cited by 129 publications

(157 citation statements)

References 32 publications

Supporting

Mentioning

155

Contrasting

Order By: Relevance

“…2 As a first step towards optimizing the function of PPBR4, we confirmed that phage displaying PPBR4 could be selected over phage bearing aPP when the two were sorted on the basis of specific DNA-binding. 19 Phage displaying either PPBR4 or its progenitor aPP were panned against magnetic beads coated with a 24 bp duplex oligonucleotide containing the 5 bp sequence recognized by PPBR4, half site CRE (hsCRE, ATGAC).…”

Section: Miniature Proteins Displayed On Phage Can Be Functionally Sementioning

confidence: 54%

“…With notable exceptions, 1 removal of these tertiary interactions destabilizes the a-helix and results in molecules that neither fold nor function in macromolecular recognition. 2 The ability to recapitulate or perhaps even improve upon the recognition properties of an a-helix within the context of a small molecule should find utility in the design of synthetic mimetics or inhibitors of protein function 3 and as new tools for proteomics research.Two fundamentally different approaches have been taken to bestow a-helical structure on otherwise unstructured peptide sequences. One approach makes use of modified amino acids or surrogates that favor helix initiation 4 or helix propagation.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…Because of its small size and stability, aPP is an excellent scaffold for protein grafting of a-helical recognition epitopes. 2 In contrast to the case for traditional protein minimization, it is easy to envision how protein grafting could stabilize almost any a-helical epitope, since the necessary stabilizing interactions can theoretically be provided by 0960-894X/01/$ -see front matter # 2001 Elsevier Science Ltd. All rights reserved. P I I : S 0 9 6 0 -8 9 4 X ( 0 1 ) 0 0 1 3 9 -1 the aPP scaffold irrespective of the tertiary complexity of the natural protein.…”

mentioning

confidence: 99%

“…2 Grafting of the GCN4 residues responsible for DNA recognition 14 onto the exterior surface of the aPP a-helix yielded the 42 amino acid peptide PPBR4. 2 PPBR4 recognized specific DNA well at 4 C, forming a complex with an equilibrium dissociation constant (K d ) of 2.3 nM.…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Chin

Grotzfeld

Fabian

et al. 2001

Bioorganic & Medicinal Chemistry Letters

Self Cite

View full text Add to dashboard Cite

Section: Miniature Proteins Displayed On Phage Can Be Functionally Sementioning

confidence: 54%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Chin

Grotzfeld

Fabian

et al. 2001

Bioorganic & Medicinal Chemistry Letters

Self Cite

View full text Add to dashboard Cite

Design and Evolution of a Miniature Bcl-2 Binding Protein

Chin

Schepartz

2001

Angew. Chem.

Self Cite

View full text Add to dashboard Cite

The design of molecules that bind protein surfaces with nanomolar affinity and high specificity presents a major challenge for chemical biologists. [1] Molecules that possess these properties have potential as therapeutic leads for protein targets [2] and new tools for proteome research. [3] We have described a general strategy for the design of miniature ahelical proteins [4] that bind DNA with high affinity and selectivity. [5] This protein grafting strategy involves identifying the a-helical residues used for DNA recognition by a natural DNA-binding protein and substituting them on the small, stable protein scaffold avian pancreatic polypeptide (aPP). [6] Grafting of the DNA contact residues of the bZIP protein GCN4 [7] followed by functional selection generated a folded miniature protein that bound DNA with exceptional affinity and specificity. [8] Here we demonstrate the versatility of protein grafting for the design of selective miniature proteinbinding proteins ( Figure 1) and report highly potent and specific ligands for human Bcl-2 and Bcl-X L . [9] Figure 1. Protein grafting and evolution of high affinity miniature protein ligands for Bcl-2 and Bcl-X L .CH 2 ), 2.42 (t, 2 H, CH 2 ), 3.48 (s, 9 H, NCH 3 ), 3.72 (2 H, NCH 2 ); FAB-MS: 276 ([M À Br] calcd 276). 1 c: 1 H NMR (CDCl 3 ): d 0.90 (t, 3 H, CH 3 ), 1.37 (14 H, CH 2 ), 1.77 (2 H, CH 2 ), 2.26 (4 H, CH 2 ), 3.48 (s, 9 H, NCH 3 ), 3.60 (2 H, NCH 2 ); FAB-MS: 276 [M À Br] (calcd 276), 634 [2 M À Br] (calcd 633).Thermally induced polymerization of 1 b, 2 a, and 2 b was carried out by heating a N 2 -purged glass tube containing a powder sample in a Kugelrohr apparatus at 170 8C for 2 h. For extraction of the included polymer from 3 b, the composite powder was suspended in aqueous hydrofluoric acid (55 wt %) at room temperature for 1 h, and N 2 was bubbled into the resulting clear solution to flush out volatile fractions.Absorption and fluorescence spectra were recorded in reflection mode on JASCO U-best V-560 and FL-777W spectrophotometers, respectively, with attachments for powder samples. Infrared spectra of KBr pellet samples were recorded on a JASCO FTIR-610 spectrophotometer. X-ray diffraction patterns were recorded on a Rigaku RINT 2400 diffractometer with Cu Ka radiation (20 kV, 50 mA) and a step size of 0.0048 at a scan speed of 18 min À1 . Optical and fluorescence micrographs were taken by an Olympus VANOX AH2-FL microscope, and excitation (395 ± 415 nm) and emission filters (b 455 nm) were used for fluorescence microscopy. Thermogravimetric analysis (TGA) was carried out on a Shimadzu TGA-50 at a heating rate of 10 8C min À1 . Transmission electron microscopy (TEM) was performed on a Hitachi H-9000UHR electron microscope operated at 300 kV.

show abstract