5MAK

GFP-binding DARPin fusion gc_R7

Summary for 5MAK

• Entry DOI: 10.2210/pdb5mak/pdb
• Descriptor: Green fluorescent protein, R7, CITRIC ACID, ... (4 entities in total)
• Functional Keywords: green fluorescent protein, designed ankyrin repeat protein, fluorescent protein
• Biological source: Aequorea victoria (Jellyfish); More
• Total number of polymer chains: 4
• Total formula weight: 118646.55

Authors

Hansen, S.,Stueber, J.,Ernst, P.,Koch, A.,Bojar, D.,Batyuk, A.,Plueckthun, A. (deposition date: 2016-11-03, release date: 2017-11-08, Last modification date: 2023-11-15)

Primary citation

Hansen, S.,Stuber, J.C.,Ernst, P.,Koch, A.,Bojar, D.,Batyuk, A.,Pluckthun, A.
Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity.
Sci Rep, 7:16292-16292, 2017

PubMed 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.

PubMed: 29176615
DOI: 10.1038/s41598-017-15711-z

Experimental method

X-RAY DIFFRACTION (2.5 Å)