title: Detection of specific protein-protein interactions in nanocages by engineering bipartite FlAsH binding sites.
abstract: Proteins that form cage-like structures have been of much recent cross-disciplinary interest due to their application to bioconjugate and materials chemistry, their biological functions spanning multiple essential cellular processes, and their complex structure, often defined by highly symmetric protein–protein interactions. Thus, establishing the fundamentals of their formation, through detecting and quantifying important protein–protein interactions, could be crucial to understanding essential cellular machinery, and for further development of protein-based technologies. Herein we describe a method to monitor the assembly of protein cages by detecting specific, oligomerization state dependent, protein–protein interactions. Our strategy relies on engineering protein monomers to include cysteine pairs that are presented proximally if the cage state assembles. These assembled pairs of cysteines act as binding sites for the fluorescent reagent FlAsH, which, once bound, provides a readout for successful oligomerization. As a proof of principle, we applied this technique to the iron storage protein, DNA-binding protein from starved cells from E. coli. Several linker lengths and conformations for the presentation of the cysteine pairs were screened to optimize the engineered binding sites. We confirmed that our designs were successful in both lysates and with purified proteins, and that FlAsH binding was dependent upon cage assembly. Following successful characterization of the assay, its throughput was expanded. A two-dimension matrix of pH and denaturing buffer conditions was screened to optimize nanocage stability. We intend to use this method for the high throughput screening of protein cage libraries and of conditions for the generation of inorganic nanoparticles within the cavity of these and other cage proteins.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Cornell
authors.forename: Jing
authors.initials: J
authors.lastname: Fu
authors.forename: Stephanie H
authors.initials: SH
authors.lastname: Newland
authors.forename: Brendan P
authors.initials: BP
authors.lastname: Orner
mesh_terms: D001665:Binding Sites; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D049329:Nanostructures / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins