title: Supramolecular Assembly of an Evolved Miniprotein Host and Fluorogenic Guest Pair.
abstract: Small-molecule-induced assembly of defined protein structures could have broad implications for the fabrication of new materials as well as biological signaling pathways. However, the design of new host-guest pairs capable of small-molecule-induced assembly in a biologically relevant context remains a significant challenge. Herein, we report a series of miniprotein hosts, evolved from the tenth type III domain of fibronectin (Fn3), that display remarkable binding affinity toward a red-shifted environment-sensitive merocyanine derivative, termed sI-Pht. Importantly, the consensus binder isolated from directed evolution experiments (6.2.18) forms a higher order assembly in response to addition of sI-Pht, as assessed by analytical ultracentrifugation. sI-Pht-induced assembly of 6.2.18 results in a 570-fold increase in fluorescence compared to free dye. This property enables the direct visualization of host-guest assemblies by fluorescence microscopy. As a demonstration, we show that supramolecular assembly of the 6.2.18-sI-Pht system can be visualized on the surface of living yeast cells. This new host-guest pair provides a tool for the potential development of new materials as well as pathway engineering. In a broader context, this work details a new design paradigm for the discovery of host-guest systems that function in the context of living cells.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
authors.forename: Bi
authors.initials: B
authors.lastname: Xu
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
authors.forename: Xinqi
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
authors.forename: Cliff I
authors.initials: CI
authors.lastname: Stains
mesh_terms: D001578:Benzopyrans / Q000737:chemistry*; D005353:Fibronectins / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D015394:Molecular Structure; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology
chemical_list: D001578:Benzopyrans; D005353:Fibronectins; D007211:Indoles; D016271:Proto-Oncogene Proteins c-myc; C548873:merocyanine