Whilst Zn2+ ions are critical regulators of many fundamental cellular processes, methods to monitor the free concentrations of these ions dynamically within living cells are presently limited. We have developed a series of genetically-encoded Förster Resonance Energy Transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range, and can readily be targeted to subcellular organelles. These sensors reveal that the free cytosolic Zn2+ concentration of fibroblasts and pancreatic islet β-cells is tightly buffered at ~400 pM, a level at least 103-fold lower than that in secretory granules.

CopC is a small soluble protein expressed in the periplasm of Pseudomonas syringae pathovar tomato as part of its copper resistance response (cop operon). Equilibrium competition reactions confirmed two separated binding sites with high affinities for Cu(I) (10(-7) > or = K(D) > or = 10(-13) M) and Cu(II) (K(D) = 10(-13(1)) M), respectively. While Cu(I)-CopC was converted cleanly by O2 to Cu(II)-CopC, the fully loaded form Cu(I)Cu(II)-CopC was stable in air. Variant forms H1F and H91F exhibited a lower affinity for Cu(II) than does the wild-type protein while variant E27G exhibited a higher affinity. Cation exchange chromatography detected each of the four different types of intermolecular copper transfer reactions possible between wild type and variant forms: Cu(I) site to Cu(II) site; Cu(II) site to Cu(I) site; Cu(I) site to Cu(I) site; Cu(II) site to Cu(II) site. The availability of an unoccupied site of higher affinity induced intermolecular transfer of either Cu(I) or Cu(II) in the presence of O2 while buffering concentrations of cupric ion at sub-picomolar levels. Crystal structures of two crystal forms of wild-type Cu(I)Cu(II)-CopC and of the apo-H91F variant demonstrate that the core structures of the molecules in the three crystal forms are conserved. However, the conformations of the amino terminus (a Cu(II) ligand) and the two copper-binding loops (at each end of the molecule) differ significantly, providing the structural lability needed to allow transfer of copper between partners, with or without change of oxidation state. CopC has the potential to interact directly with each of the four cop proteins coexpressed to the periplasm.

Nature uses bottom-up approaches for the controlled assembly of highly ordered hierarchical structures with defined functionality, such as organelles, molecular motors, and transmembrane pumps. The field of bionano-technology draws inspiration from nature by utilizing biomolecular building blocks such as DNA, proteins, and lipids, for the (self-) assembly of new structures for applications in biomedicine, optics, or electronics. Among the toolbox of available building blocks, proteins that form cage-like structures, such as viruses and virus-like particles, have been of particular interest since they form highly symmetrical assemblies and can be readily modified genetically or chemically both on the outer or inner surface. Bacterial encapsulins are a class of nonviral protein cages that self-assemble in vivo into stable icosahedral structures. Using teal fluorescent proteins (TFP) engineered with a specific native C-terminal docking sequence, we report the molecular self-sorting and selective packaging of TFP cargo into bacterial encapsulins during in vivo assembly. Using native mass spectrometry techniques, we show that loading of either monomeric or dimeric TFP cargo occurs with unprecedented high fidelity and exceptional loading accuracy. Such self-assembling systems equipped with self-sorting capabilities would open up exciting opportunities in nanotechnology, for example, as artificial (molecular storage or detoxification) organelles or as artificial cell factories for in situ biocatalysis. ■ INTRODUCTION Nature assembles small building blocks into highly sophisticated architectures such as DNA helices, viruses, membrane pumps, and organelles. Hierarchical assemblies that mimic their complexity have been of increasing interest for applications in nanotechnology and synthetic biology. For example, icosahe-dral virus-like structures have been shown to be highly useful for the encapsulation of a range of functional materials (i.e., proteins, polymers, nanoparticles, and inorganic complexes) for potential applications in nanomedicine, nanoelectronics, biomedical imaging, and catalysis. 1 In recent years, the library of engineered (synthetic and biological) assemblies has expanded tremendously, and there is now a growing trend to engineer inherently interacting proteins that can spontaneously self-assemble in vitro into complex structures. 2 In the reported cases, encapsulation of functional cargo is controlled by either creating fusion proteins or by introducing favorable electro-static, hydrogen bonding, or hydrophobic interactions between the functional cargo and the protein scaffold. 3 However, in nature, this entire cargo loading and self-assembly process occurs in the crowded environment of the cell, yet does so with extremely high efficiency, and in the presence of multiple competitors. This high fidelity selectivity and affinity observed in biological systems is often termed self-sorting, the ability to identify self (desired cargo) from nonself (undesired cargo), and is extremely challenging to engineer...

We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318 -3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA. IMPORTANCECowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of such a mechanism by demonstrating that efficient assembly is initiated by the formation of a disordered protocapsid complex whose stoichiometry is governed by electrostatics (charge matching of the anionic RNA and the cationic N termini of the CP).