title: Rewiring Multidomain Protein Switches: Transforming a Fluorescent Zn(2+) Sensor into a Light-Responsive Zn(2+) Binding Protein.
abstract: Protein-based sensors and switches provide attractive tools for the real-time monitoring and control of molecular processes in complex biological environments. Fluorescent sensor proteins have been developed for a wide variety of small molecules, but the construction of genetically encoded light-responsive ligand binding proteins remains mostly unexplored. Here we present a generic approach to reengineer a previously developed FRET-based Zn(2+) sensor into a light-activatable Zn(2+) binding protein using a design strategy based on mutually exclusive domain interactions. These so-called VividZn proteins consist of two light-responsive Vivid domains that homodimerize upon illumination with blue light, thus preventing the binding of Zn(2+) between two Zn(2+) binding domains, Atox1 and WD4. Following optimization of the linker between WD4 and the N-terminus of one of the Vivid domains, VividZn variants were obtained that show a 9- to 55-fold decrease in Zn(2+) affinity upon illumination, which is fully reversible following dark adaptation. The Zn(2+) affinities of the switch could be rationally tuned between 1 pM and 2 nM by systematic variation of linker length and mutation of one of the Zn(2+) binding residues. Similarly, introduction of mutations in the Vivid domains allowed tuning of the switching kinetics between 10 min and 7 h. Low expression levels in mammalian cells precluded the demonstration of light-induced perturbation of cytosolic Zn(2+) levels. Nonetheless, our results firmly establish the use of intramolecular Vivid dimerization as an attractive light-sensitive input module to rationally engineer light-responsive protein switches based on mutually exclusive domain interactions.
authors.affiliation: Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology , P.O. Box 513, 5600 MB Eindhoven, The Netherlands.
authors.forename: Stijn J A
authors.initials: SJ
authors.lastname: Aper
authors.affiliation: Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology , P.O. Box 513, 5600 MB Eindhoven, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000235:genetics; D000072417:Protein Domains; D015202:Protein Engineering / Q000379:methods*; D015032:Zinc / Q000032:analysis*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; C434479:citrine protein, bacteria; C017800:zinc-binding protein; D005182:Flavin-Adenine Dinucleotide; D015032:Zinc
keywords: Vivid; fluorescent sensor; light switching; metal binding; optogenetics; protein engineering