Assembly-pathway regulation dictates pH-responsive actuation in the R-body protein machinery

Refractile bodies (R-bodies) are protein assemblies that form tightly rolled morphologies in cells and undergo large-scale extension into long spirals in response to environmental stimuli. The type 51 R-body, the focus of this study, is assembled from four Reb proteins and undergoes a rapid repeatable ~50-fold extension. Although R-bodies were first described more than 70 years ago, it has remained unclear how RebC and RebD contribute to the formation and function of this four-protein machinery even though RebA and RebB have been proposed as its major components. We characterized the wild-type R-body (Rb_WT) and a series of reb gene knockout mutants by combining in-cell, biochemical analyses, small-angle X-ray scattering (SAXS) and attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. We found that RebD is incorporated into Rb_WT as a minor component, while RebC is not detectably incorporated into the final assembly. Mutants lacking either RebA or RebB still form roll-like assemblies but lack pH-dependent extension. In contrast, mutants lacking RebC and/or RebD exhibit a substantially lower propensity for roll formation and instead undergo off-pathway aggregation with increased β-sheet content. SAXS analyses further indicate that roll-like morphology does not ensure formation of the ordered lamellar architecture characteristic of functional Rb_WT. Thus, pH-responsive R-body actuation is dictated not simply by the major proteins that constitute the final architecture, but by a regulated assembly pathway that builds the ordered lamellar architecture required for actuation. These findings establish assembly-pathway regulation as a key principle for constructing dynamic protein architectures capable of stimuli-responsive mechanical actuation.