Tuning microscale, retractable, membrane-breaking protein needles

Abstract

The refractile (R) bodies found in Caedibacter taeniospiralis, a bacterial endosymbiont of Paramecium tetraurelia, are large, polymeric protein structures that can switch between two conformations. At cytoplasmic pH, they resemble coiled ribbons of protein 500nm in diameter. At low pH, they extend to form hollow needles up to 20 microns long. They can be expressed heterologously from an operon containing four short open reading frames and can function in vitro in diverse buffer conditions.\n\nIn this study, R bodies purified from Escherichia coli were found to be capable of undergoing many consecutive extension-contraction cycles. Furthermore, the solubility of R bodies, which can easily be interpreted by eye, was found to correlate with their extension state. This macroscopic phenotype was used to develop a quantitative, high-throughput assay for R body state, enabling a visual screen of R body mutants defective in extension. The role of specific amino acids in extension was determined, and this information was used to construct rationally-designed mutants tailored to extend at higher pH. Furthermore, R bodies were able to rupture E. coli spheroplasts to release soluble proteins across lipid bilayers. Taken together, these results show that R bodies act as tunable, pH-actuated pistons suitable for a variety of membrane-breaking applications.