PDBsum entry 4j8y

De novo protein

PDB id: 4j8y

Contents

Protein chain

157 a.a.

Waters ×167

PDB id:

4j8y

Name:

De novo protein

Title:

E3_5 darpin d77s mutant

Structure:

Darpin_e3_5_d77s. Chain: a. Engineered: yes

Source:

Synthetic construct. Organism_taxid: 32630. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.70Å R-factor: 0.173 R-free: 0.207

Authors:

M.A.Seeger,M.G.Gruetter

Key ref:

M.A.Seeger et al. (2013). Design, construction, and characterization of a second-generation DARP in library with reduced hydrophobicity. Protein Sci, 22, 1239-1257. PubMed id: 23868333 DOI: 10.1002/pro.2312

Date:

15-Feb-13 Release date: 14-Aug-13

Protein chain

No UniProt id for this chain

Struc: 157 a.a.

DOI no: 10.1002/pro.2312

Protein Sci 22:1239-1257 (2013)

PubMed id: 23868333

Design, construction, and characterization of a second-generation DARP in library with reduced hydrophobicity.

M.A.Seeger, R.Zbinden, A.Flütsch, P.G.Gutte, S.Engeler, H.Roschitzki-Voser, M.G.Grütter.

ABSTRACT

Designed ankyrin repeat proteins (DARPins) are well-established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin-target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase-7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.