title: Design of functional ferritin-like proteins with hydrophobic cavities.
abstract: Ferritin four-helix bundle subunits self-assemble to create a stable multimer with a large central hydrophilic cavity where metal ions bind. To explore the versatility of this reaction vessel, computational design was used to generate cavities with increasingly apolar surface areas inside a dodecameric ferritin-like protein, Dps. Cavity mutants, in which as many as 120 surface accessible hydrophilic residues were replaced with hydrophobic amino acids, were shown to still assemble properly using size-exclusion chromatography and dynamic light scattering measurements. Wild-type Dps exhibited highly cooperative subunit folding and assembly, which was monitored by changes in Trp fluorescence and UV circular dichroism. The hydrophobic cavity mutants showed distinctly less cooperative unfolding behavior, with one mutant forming a partially assembled intermediate upon guanidine denaturation. Although the stability of Dps to such denaturation decreased with increasing apolar surface area, all proteins exhibited high melting temperatures, T(m) = 74-90 degrees C. Despite the large number of mutations, near-native ability to mineralize iron was maintained. This work illustrates the versatility of the ferritin scaffold for engineering large protein cavities with novel properties.
authors.affiliation: Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, USA.
authors.forename: Joe
authors.initials: J
authors.lastname: Swift
authors.forename: William A
authors.initials: WA
authors.lastname: Wehbi
authors.forename: Brenna D
authors.initials: BD
authors.lastname: Kelly
authors.forename: Xiaoran Fu
authors.initials: XF
authors.lastname: Stowell
authors.forename: Jeffery G
authors.initials: JG
authors.lastname: Saven
authors.forename: Ivan J
authors.initials: IJ
authors.lastname: Dmochowski
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002853:Chromatography, Liquid; D019295:Computational Biology; D017076:Computer-Aided Design; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D007501:Iron / Q000737:chemistry; D008958:Models, Molecular; D016296:Mutagenesis; D011489:Protein Denaturation / Q000187:drug effects; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C083911:DPS protein, Bacteria; D005293:Ferritins; D007501:Iron; D019791:Guanidine