Coiled-coil domains are attractive modular components for assembling individual protein subunits into higher order structures because they can be designed de novo with well-defined oligomerization states, topologies, and dissociation energies. However, the utility of coiled-coil designs as plug-and-play components for synthetic biology applications depends critically on them robustly maintaining their oligomerization states when fused to larger proteins of interest. Here, we investigate the ability of a series of wellcharacterized de novo-designed parallel coiled coils, with oligomerization states ranging from dimer to pentamer, to mediate the oligomerization of a model monomeric protein, green fluorescent protein (GFP). Six coiled-coil GFP fusion proteins were initially constructed and their oligomerization states investigated using size exclusion chromatography, analytical ultracentrifugation, and native mass spectrometry. Somewhat surprisingly, only two of these initial designs adopted their intended oligomerization states. However, with minor refinements, the intended oligomerization states of two of the four other constructs could be achieved. Parameters found to influence the oligomerization state of the GFP fusions included the number of heptad repeats and the length of the linker sequence separating GFP from the coiled coil. These results demonstrate that even for stable, well-designed coiled coils, the oligomerization state is subject to unanticipated changes when connected to larger protein components. Therefore, although coiled coils can be successfully used as components in protein designs their ability to achieve the desired oligomerization state requires experimental verification.

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 assembly of individual protein subunits into large-scale structures is important in many biological contexts. Proteins may assemble into geometrical cages or extended lattices that are characterized by a high degree of symmetry; examples include viral capsids and bacterial S-layers. The precisely defined higher order structure exhibited by these assemblies has inspired efforts to design such structures de novo by applying the principles of symmetry evident in natural protein assemblies. Here we discuss progress towards this goal and also examples of natural protein cages and lattices that have been engineered to repurpose them towards a diverse range of applications in materials science and nano-medicine.

The cover picture shows the application of symmetry principles to assembling proteins into geometrical cages. In their communication, E. N. G. Marsh et al. explain how they used a small, trimeric, coiled-coil domain to assemble a trimeric enzyme into a well-defined tetrahedral cage. This was achieved by genetically fusing the coiled-coil domain to the C terminus of the enzyme protein through a flexible polyglycine linker sequence—the coiled-coil acts as a molecular “twist-tie” to hold the cage together. Other than optimizing the strength of the coiled-coil interaction and the length of the glycine linker, no further constraints were needed to assemble the desired tetrahedral cage in high yield. This methodology provides a flexible and modular symmetry-based approach to assembling polyhedral protein cages that can be expanded to use de novo-designed coiled coils as off-the-shelf components for protein assembly. More details can be found in the communication by E. N. G. Marsh et al. on page 1888 in Issue 19, 2017 (DOI: 10.1002/cbic.201700406).

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-symmetriccoiled-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.

Significance The ability to organize biological molecules into new hierarchical forms represents an important goal in synthetic biology. However, designing new quaternary interactions between protein subunits has proved technically challenging and has generally required extensive redesign of protein−protein interfaces. Here, we demonstrate a conceptually simple way to assemble a protein into a well-defined geometric structure that uses coiled-coil sequences as “off-the-shelf” components. This approach is inherently modular and adaptable to a wide range of proteins and symmetries, opening up avenues for the construction of biological structures with diverse geometries and wide-ranging functionalities.

: 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-symmetriccoiled-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.

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.

In dieser Zuschrift charakterisierten die Autoren die durch cyanobakterielle Aldehyd-Decarbonylase (cAD) katalysierte Decarbonylierung als Sauerstoff-unabhängigen Prozess. Weitere Experimente haben sie nun zu dem Schluss geführt, dass Sauerstoffspuren im Puffer als Ursache für die beobachtete Aktivität nicht ausgeschlossen werden können. Die Schwierigkeiten bei der Ermittlung der Sauerstoffabhängigkeit der betrachteten Reaktion gehen unter anderem auf die sehr geringe Aktivität des Enzyms unter aeroben wie anaeroben Bedingungen zurück. Routinemäßig zum Entfernen von Sauerstoff bei biochemischen Reaktionen eingesetzte Reagentien wie Natriumdithionit oder Systeme wie Glucose-Oxidase/Glucose und Protocatechuat-Dioxygenase/Protocatechuat verminderten die Aktivität von cAD unter den in der Zuschrift angewendeten Testbedingungen selbst in großem Überschuss nicht. Diese Beobachtung stützt zwar die ursprüngliche Behauptung, Sauerstoff sei nicht an der Reaktion beteiligt, doch Tests in einer Luftausschlusskammer bei sehr niedrigen Sauerstoffkonzentrationen (unter 0.5 ppm, was bei dem ursprünglichen Experiment nicht möglich war) ergaben sehr geringe Aktivitäten. Infolge dieser Unstimmigkeit kann die Beteiligung von molekularem Sauerstoff an der cAD-katalysierten Reaktion nicht zweifelsfrei ausgeschlossen werden. Alle sonstigen Daten und Schlussfolgerungen des Beitrags bleiben von dieser Beobachtung unberührt, der vorgeschlagene Mechanismus der Enzymaktivität muss aber überprüft werden.