We report thermally triggered self-assembly of folded proteins into vesicles that incorporates globular proteins as building blocks. Leucine zipper coiled coils were combined with either globular proteins or elastin-like polypeptides as recombinant fusion proteins, which form "rod-coil" and "globule-rod-coil" protein complex amphiphiles. In aqueous solution, they self-assembled into hollow vesicles via temperature-responsive inverse phase transition. The characteristic of the protein vesicle membranes enables preferential encapsulation of simultaneously formed protein coacervate. Furthermore, the type of encapsulated cargo extends to small molecules and nanoparticles. Our approach offers a versatile strategy to create protein vesicles as vehicles with biological functionality.

Synthetic protein assemblies that adopt programmed shapes would support many applications in nanotechnology. We used a rational design approach that exploits the modularity of orthogonally interacting coiled coils to create a self-assembled protein nanotriangle. Coiled coils have frequently been used to construct nanoassemblies and materials, but rarely with successful prior specification of the resulting structure. We designed a heterotrimer from three pairs of heterodimeric coiled coils that mediate specific interactions while avoiding undesired crosstalk. Non-associating pairs of coiled-coil units were strategically fused to generate three chains that were predicted to preferentially form the heterotrimer, and a rational annealing process led to the desired oligomer. Extensive biophysical characterization and modeling support the formation of a molecular triangle, which is a shape distinct from naturally occurring supramolecular nanostructures. Our approach can be extended to design more complex nanostructures using additional coiled-coil modules, other protein parts, or templated surfaces.

Mimicry of biomineralization is an attractive strategy to fabricate nanostructured hybrid materials. While biomineralization involves processes that organize hybrid clusters into complex structures with hierarchy, arrangement of artificial components in biomimetic approaches has been challenging. Here, we demonstrate self-assembly of hierarchically structured porous supraparticles from protein-inorganic hybrid flower-shaped (FS) nanoparticle building blocks. In our strategy, the FS nanoparticles self-assemble via high valency interactions in combination with interfacial adsorption and compression. The flower-like shape directed robust assembly of the FS nanoparticles into chain-like clusters in solution, which were further assembled into spherical supraparticles during rotation of FS nanoparticle solution. Continuously expanding and contracting the air-water interface during rotation catalyzed assembly of FS nanoparticle clusters, indicating that adsorption and compression of the building blocks at the interface were critical. The resulting supraparticles contain hierarchical pores which are translated from the structural characteristics of individual FS nanoparticle building blocks. The protein-inorganic supraparticles are protein-compatible, have large surface area, and provide specific affinity recognition for robust protein immobilization. A variety of functional proteins could be immobilized to the porous supraparticles, making it a general platform that could provide benefits for many applications.

Highlights d One-photon calcium imaging of brain activity can suffer from neuropil crosstalk d Targeting GCaMPs to the cell body reduces neuropil crosstalk d One-photon imaging of somatic GCaMP reduces artifactual spikes and correlations d Somatic GCaMPs can be used in multiple species, such as mice and zebrafish

Therapeutic proteins require controlled delivery to the target tissue to maintain the local concentration over a prolonged period of time. A common approach is to delay protein release with a hydrogel matrix, which provides affinity interactions or diffusive barriers. [1] Physical entrapment of proteins in a matrix allows sustained release due to free volume, hydrodynamic drag, and obstruction effects. [2] Affinity-based approaches exploit specific binding interactions between natural [3] or engineered binding ligands [4] attached to the hydrogel matrix and therapeutic proteins. However, hydrogel-based approaches inherently involve relatively large amounts of carrier material compared to the entrapped protein therapeutic. Toxicity concerns may arise from synthetic crosslinkers or initiators used in polymerization, [5] or modification of biological polymers. [6] Biocompatibility issues, such as the foreign body response, may be present at the interface between tissue and materials. [7] Thus, a strategy that uses no or a minimal amount of carrier material is ideal, to eliminate these challenges. Herein, we demonstrate that the concept of "carrier-free" can be realized by adopting a different physical principle, protein self-assembly.Self-assembly has been exploited as a useful tool to fabricate ordered structures from peptides [8] or proteins. [9] While much effort has been focused on fabrication of selfassembled matrices, [10] recent studies reported that protein properties, including binding avidity, [11] bioactivity, [12] and stability, [13] are modulated by protein self-assembly. In this study, we describe a self-assembly system in which proteins self-control their molecular transport in a model extracellular matrix (ECM). Engineered protein building blocks spontaneously self-assemble into particles in the ECM, become trapped, and dissociate to be released at a controlled rate. This protein self-assembly is mediated by temperatureresponsive coacervation and dissociation, and by specific binding of high-affinity protein motifs under physiological conditions. Since the ECM selectively regulates microscopic motion of objects depending on their size, [14] the diffusion of protein building blocks is modulated as they form particles under non-equilibrium conditions.The system is built from two different di-block fusion proteins. The first component (mCherry-Z E ) is constructed from a globular fluorescent protein mCherry, [15] which serves as a model for a therapeutic protein, and a glutamic acid-rich leucine zipper (Z E ). An arginine-rich leucine zipper (Z R ) and elastin-like polypeptide (ELP) comprise the second component (Z R -ELP) [16] (Figure 1 a). ELP is composed of a pentapeptide repeat derived from tropoelastin, which undergoes a temperature-responsive inverse phase transition from soluble to coacervate phase. [17] The leucine zipper motifs Z E and Z R form heterodimeric a-helical coiled-coils with a dissociation constant, K D % 10 À15 m. [18] The fusion proteins were produced separately by using bacterial ...

The strategies of pathogens to evade the human immune system are highly sophisticated and modulate a variety of inflammatory pathways.

Highlights d Clustering fluorescent sensors at points in cells enables many to be imaged at once d Modular reagent design allows existing sensors to be easily adapted to cluster d Such ''signaling reporter islands'' (SiRIs) are safe and robust in cells and in vivo d SiRIs reveal relationships between components of signal transduction networks

Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their ability to simultaneously capture the dynamics of hundreds of neurons across large fields of view, at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk between cell bodies and the surrounding neuropil, resulting in decreased signal-to-noise and artifactual correlations of neural activity. Here, we address this problem by engineering cell body-targeted variants of the fluorescent calcium indicator GCaMP6f. We screened fusions of GCaMP6f to both natural as well as engineered peptides, and identified fusions that localized GCaMP6f to within approximately 50 microns of the cell body of neurons in live mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP6f in dense neural circuits reported fewer artifactual spikes from neuropil, increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators increases neuronal signal fidelity and may facilitate even greater usage of simple, powerful, one-photon methods of population imaging of neural calcium dynamics.