Design and Evolution of a Miniature Bcl-2 Binding Protein

Abstract

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.

Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell Surface

“…Neither of the isoleucine residues in positions 81 and 85 is strongly retained, and Ile85 is always replaced with phenylalanine. The I81F mutation was previously observed in phage display studies to isolate miniature protein/Bak chimeras with high affinity to Bcl-2 38. We observe the I81F mutation once, but leucine is more strongly preferred in position 81 in the clones found in our library.…”

Limiting Assumptions in the Design of Peptidomimetics

“…a-and 3 10 -helices ( Fig. 11) The Hamilton [Orner et al, 2001;Ernst et al, 2003] and Schepartz [Chin and Schepartz, 2001;Rutledge et al, 2002Rutledge et al, , 2003] groups pioneered synthetic efforts to mimic a-helical recognition elements with small, hybridized proteins and nonpeptides which inspired others to consider alternative strategies. Several reviews of the various approaches to stabilizing/ mimicking helical peptides have appeared [Garner and Harding, 2007;Henchey et al, 2008;Pelay-Gimeno et al, 2015].…”

Small Scaffolds, Big Potential: Developing Miniature Proteins as Therapeutic Agents

“…Bcl-2 is an antiapoptotic mitochondrial membrane protein that functions along the intrinsic apoptosis pathway (Cory et al, 2003). Helical pro-apoptotic proteins, such as Bak, bind and neutralize anti-apoptotic Bcl-2 proteins on the mitochondrial membrane, facilitating the release of cytochrome c. In 2001, Chin et al used phage display to graft residues from the helical Bcl-2 interaction domain of Bak required for Bcl-2 recognition to the ahelix of aPP (Chin and Schepartz, 2001). One construct, designated PPBH3-1, adopted a stable aPP-like fold and bound Bcl-2 with a K d of 52 nM.…”