Abstract
Self-assembly is a convenient process to arrange complex biomolecules into large hierarchically ordered structures. Electrostatic attraction between the building blocks is a particularly interesting driving force for the assembly process, as it is easily tunable and reversible. Large biomolecules with high surface charge density, such as proteins and protein cages, are very promising building blocks due to their uniform size and shape. Assemblies of functional molecules with well-defined nanostructures have wide-ranging applications but are difficult to produce precisely by synthetic methods. Furthermore, obtaining highly ordered structures is an important prerequisite for X-ray structure analysis. Here we show how negatively charged ferritin and viral protein cages can adopt specific cocrystal structures with supercharged cationic polypeptides (SUPs, K72) and their recombinant fusions with green fluorescent protein (GFP-K72). The cage structures and recombinant proteins self-assemble in aqueous solution to large ordered structures, where the structure morphology and size are controlled by the ratio of oppositely charged building blocks and the electrolyte concentration. Both ferritin and viral cages form cocrystals with face centered cubic structure and lattice constants of 14.0 and 28.5 nm, respectively. The crystals are porous and the cationic recombinant proteins occupy the voids between the cages. Such systems resemble naturally occurring occlusion bodies and may serve as protecting agents as well as aid the structure determination of biomolecules by X-ray scattering.
Highlights
M imicking the highly evolved functionalities of native biomolecules has been in the focus of research efforts, especially over the past decade.[1]
Volume-average size distribution profiles of chlorotic mottle virus (CCMV) with (e) K72, (f) green fluorescent protein (GFP)-K72, and apoferritin from Pyrococcus f uriosus (aFT) with (g) K72, (h) GFP-K72 at different stages of titration and b), respectively). (i) Agarose gel electrophoresis mobility shift assay (EMSA) demonstrating the effect of increasing K72 and GFP-K72 concentration on the electrophoretic mobility of CCMV
We have previously shown that positively charged avidin proteins and negatively charged protein cages can form ordered structures through electrostatic self-assembly.[30]
Summary
Titrated with K72 and GFP-K72, (c) electrolyte (NaCl) induced disassembly of the CCMV complexes, and (d) electrolyte induced disassembly of the aFT complexes. The formed structures were disassembled by titration with aqueous sodium chloride (NaCl) solution, as a sufficiently high electrolyte concentration screened the electrostatic interactions between K72 and the protein cages Both CCMV complexes disassembled uniformly when titrated with NaCl solution (Figure 2c). In the case aFT, a steady decrease in the count rate was observed for K72 complex, but aFT−GFP-K72 underwent an increase in the count rate at the beginning of the NaCl titration (Figure 2d) This indicates that the latter structures did not disassemble uniformly throughout the solution but broke first into numerous smaller assemblies. The complexes adopted fcc packed crystalline morphology except for the aFT−GFP-K72 complex, which had an amorphous structure This is most likely due to steric hindrance caused by GFP and is not present in CCMV complex because the cavities between the protein cages are large enough to house GFP.
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