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 ve

SUBMITTER: Hansen S 

PROVIDER: S-EPMC5701241 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature