Fig. 2: KaiB–KaiC complexes crosslink colloids with high specificity in a phosphorylation-dependent manner.

Fluorescence microscopy images of suspensions of 1-µm diameter colloids taken at 1 h (top), 7 h (middle), and 28 h (bottom) after mixing with KaiC mutants that are frozen in A non-binding (pT) or B binding (pS) states show substantial clustering and assembly of pS-colloids over time that is absent for pT-colloids. Fluorescence microscopy images of suspensions of colloids of 2 µm (C) and 6 µm (D) diameter in the presence of pT and pS KaiC proteins show that timed aggregation, dependent on the phosphorylation state of KaiC, is preserved for different sizes of colloids. The concentrations of colloids, proteins, and reagents, as well as imaging parameters, are identical to those in (A, B). E Images of a suspension of 1-µm diameter colloids undergoing sedimentation in a 12 mm long capillary over 28 h in the presence of pT (left) and pS (right) show that pS-colloids sediment more quickly, as indicated by dark regions extending further down the images. The time that each image is captured is listed at the top. F The same suspension parameters as in (A, B) but without KaiC (including only KaiB and b-KaiB) show minimal clustering over the course of 1 h (top) to 28 h (bottom), demonstrating that the KaiB–KaiC complex formation is essential to the colloidal self-assembly shown in (A–E). G Suspensions of streptavidin-coated colloids, with identical conditions to those in (A, B), but with Kai proteins replaced with alternative biotinylated constructs that could, in principle, crosslink streptavidin-coated colloids: (left) 1 kDa biotin-PEG-biotin with 1 biotin on each end, (middle) 20 kDa biotin-PEG-biotin with 1 biotin on each end, and (right) biotin-BSA with 8-16 biotins. The molarity of PEG and BSA matched the KaiC molarity used in (A–E), and minimal clustering is observed from 1 h (top) to 28 h (bottom), demonstrating that the effect shown in (A–F) is unique to the KaiB–KaiC binding interaction.