Just add water: Structurally, cyanobacterial aldehyde decarbonylases are members of the non‐heme diiron oxygenase family of enzymes. However, the enzyme catalyzes the hydrolysis of aliphatic aldehydes to alkanes and formate (see scheme), in an oxygen‐independent reaction. This unusual and chemically difficult reaction most likely involves free radical intermediates.

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 C 3 -symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C 4 -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 C 4 and C 3 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.coiled coils | protein design | native mass spectrometry | analytical ultracentrifugation | cryoelectron microscopy

The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and typically requires extensive redesign of protein-protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiled-coil domains to assemble proteins into higher-order structures. Here, we demonstrate the assembly of a trimeric enzyme into a well-defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiled-coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiled-coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.

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-(1) and A-(2), 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 C 3 symmetry. The coiledcoil 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-(1) and A-(2) 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-(1) and A-(2) 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.

Einfach Wasser zugeben: Mit ihrer Struktur gehören cyanobakterielle Aldehyd‐Decarbonylasen zur Enzymfamilie der Nichthäm‐Dieisen‐Oxygenasen – allerdings katalysiert das Enzym, in einer Sauerstoff‐unabhängigen Reaktion, die Hydrolyse von aliphatischen Aldehyden zu Alkanen und Formiat (siehe Schema). Die ungewöhnliche und chemisch schwierige Reaktion läuft höchstwahrscheinlich unter Beteiligung freier Radikale ab.