title: Characterization of a highly flexible self-assembling protein system designed to form nanocages.
abstract: The design of proteins that self-assemble into well-defined, higher order structures is an important goal that has potential applications in synthetic biology, materials science, and medicine. We previously designed a two-component protein system, designated A-(+) and A-(-), in which self-assembly is mediated by complementary electrostatic interactions between two coiled-coil sequences appended to the C-terminus of a homotrimeric enzyme with C3 symmetry. The coiled-coil sequences are attached through a short, flexible spacer sequence providing the system with a high degree of conformational flexibility. Thus, the primary constraint guiding which structures the system may assemble into is the symmetry of the protein building block. We have now characterized the properties of the self-assembling system as a whole using native gel electrophoresis and analytical ultracentrifugation (AUC) and the properties of individual assemblies using cryo-electron microscopy (EM). We show that upon mixing, A-(+) and A-(-) form only six different complexes in significant concentrations. The three predominant complexes have hydrodynamic properties consistent with the formation of heterodimeric, tetrahedral, and octahedral protein cages. Cryo-EM of size-fractionated material shows that A-(+) and A-(-) form spherical particles with diameters appropriate for tetrahedral or octahedral protein cages. The particles varied in diameter in an almost continuous manner suggesting that their structures are extremely flexible.
authors: Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109.
authors: Dustin P
authors: DP
authors: Patterson
authors: Min
authors: M
authors: Su
authors: Titus M
authors: TM
authors: Franzmann
authors: Aaron
authors: A
authors: Sciore
authors: Georgios
authors: G
authors: Skiniotis
authors: E Neil G
authors: EN
authors: Marsh
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D020285:Cryoelectron Microscopy; D049329:Nanostructures / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D055672:Static Electricity
chemical_list: D010455:Peptides; D011506:Proteins
keywords: analytical ultracentrifugation; coiled-coil; cryo-electron microscopy; protein cages; self-assembly; symmetry