Abstract 1525 Structural and functional diversity of calcium-triggered contractile proteins in ciliates

Ciliated protozoa use conventional ATP-powered cytoskeletal motors like myosins, dyneins, and kinesins for motility and intracellular transport. However, they also utilize novel cytoskeletal motors that do not require ATP to generate force. These motors, triggered by calcium, drive contractile processes, some of which are ultrafast. We have identified candidates for three distinct kinds of calcium-dependent, ATP-independent motor proteins in the ciliated protozoa Tetrahymena (Tcb2), Spirostomum (centrin), and Zoothamnium (spasmin). In each case, the contractile machinery comprises an EF-hand calcium-binding protein associated with an extended alpha-helical scaffold protein. Models of the individual proteins and their complexes were generated using AlphaFold. These models suggested testable mechanisms for force generation by these contractile assemblies. Genes encoding these ciliate proteins were synthesized, and plasmids were engineered to co-express each calcium-binding protein with its scaffold protein after transformation into E. coli. The co-expressed contractile proteins were purified from bacterial lysates using immobilized metal affinity chromatography. The purified, concentrated proteins form filamentous networks responsive to calcium ions. While these proteins share some similarities in structure and function, they differ in how they assemble and respond to calcium, as might be expected from the different structures they form in living cells. In addition to reconstituting the contractility of these systems using purified proteins, we are also beginning to analyze the component proteins and their assemblies using X-ray crystallography, NMR spectroscopy, and cryoelectron microscopy to gain more insight into their force-generating mechanisms. This research was supported by NSF grant award #2313727: "Collaborative Research: Understanding and controlling force generation by a centrin-based contractile system." It has also been funded by grants from Drake University and funds provided with the appointment of JH as the Marshall and Judith Flapan Professor of Biology.