title: Flexible, symmetry-directed approach to assembling protein cages.
abstract: The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Aaron
authors.initials: A
authors.lastname: Sciore
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Min
authors.initials: M
authors.lastname: Su
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Philipp
authors.initials: P
authors.lastname: Koldewey
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Eschweiler
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Kelsey A
authors.initials: KA
authors.lastname: Diffley
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Brian M
authors.initials: BM
authors.lastname: Linhares
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Brandon T
authors.initials: BT
authors.lastname: Ruotolo
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109; Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109; Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109.
authors.forename: James C A
authors.initials: JC
authors.lastname: Bardwell
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109;
authors.forename: Georgios
authors.initials: G
authors.lastname: Skiniotis
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109; Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109; nmarsh@umich.edu.
authors.forename: E Neil G
authors.initials: EN
authors.lastname: Marsh
mesh_terms: D000595:Amino Acid Sequence; D020285:Cryoelectron Microscopy; D013058:Mass Spectrometry / Q000379:methods; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D050813:Octamer Transcription Factor-2 / Q000737:chemistry / Q000648:ultrastructure; D050814:Octamer Transcription Factor-3 / Q000737:chemistry / Q000648:ultrastructure; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D050813:Octamer Transcription Factor-2; D050814:Octamer Transcription Factor-3; D011506:Proteins
keywords: analytical ultracentrifugation; coiled coils; cryoelectron microscopy; native mass spectrometry; protein design