Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

Abstract

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.

DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

“…Anti-mTFP1 DARPin 1238_E11 and DARPin 1238_G01 differ in 13 out of 21 selected library residues (Supplementary Figure S1A), suggesting alternative binding epitopes, in line with the ELISA experiments (Figure 1C). Recently, the structures of the complexes of GFP with DARPin 3G61 (PDB ID: 5MAD; Supplementary Figure S1D) and DARPin 3G124nc (PDB ID: 5MA6; Supplementary Figure S1E) were determined (Hansen et al, 2017). GFP and mTFP1 show 27% identity and 1.4 Å C α rmsd.…”

“…0.45 Å), minor differences are only observed for the two N-Cap helices. (D) Superposition of the mTFP1/DARPin 1238_E11 and the GFP/ DARPin 3G61 complex (PDB ID: 5MAD) (Hansen et al, 2017). mTFP1 (teal), DARPin 1238_E11 (grey), GFP (green) and anti-GFP DARPin 3G61 (rainbow coloured, from N-terminus blue to C-terminus red) are shown in cartoon representation.…”

Advances in targeted degradation of endogenous proteins

“…Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. [122], C-Myc [123]; LMO2 [124]; endothelial and epithelial kinase (Etk) [125]; protein kinase Cε [126]; CapG [127]; RAS [128]; caspase-3 [129]; Bax [130]; hnRNP-K [131]; L-plastin [132][133][134]; fascin [135]; cortactin [135]; gelsolin [136]; huntington [137,138]; α-synuclein [139]; β-catenin [140][141][142]; nuclear poly(A)-binding protein 1 (PABPN1) [143]; GFP [144], ASC [48] Monobody β-Catenin [145]; Abl (SH2 domain) [111,146]; SUMO1 [111,147,148]; Aurora A [149]; WDR5 [150]; H/K-RAS [151]; GFP [111]; Erk-2 [152]; ubiquitin [110] DARPIN GFP [153]; tubulin [154]; caspase-2 [155,156]; caspase-7 [155,…”

The Role of Interleukin-37 in the Pathogenesis of Allergic Diseases

“…Allergic rhinitis (AR), which is characterized by chronic inflammation in the upper respiratory tract [7], can significantly reduce a patient's quality of life by affecting his/her sleep and performance status [2]. Atopic dermatitis (AtD) is a multifactorial inflammatory skin disease which may be partly due to genetically caused impairment of the skin barrier function [8,9].…”