Detergent-based preparation influences TMEM16F assembly and scramblase activity.

Transmembrane protein 16F (TMEM16F) is a Ca2+-dependent phospholipid scramblase that mediates phosphatidylserine externalization in various physiological processes, including blood coagulation. Although previous cryo-electron microscopy studies have established the homodimeric structure of TMEM16F and provided important structural insights, the mechanisms underlying substrate translocation and functional regulation remain incompletely understood. Here, we conducted a detailed in vitro analysis of TMEM16F using a reconstituted liposome system to elucidate its regulatory mechanisms. We found that the detergents used during protein purification strongly affect both the scramblase activity and the oligomeric state of TMEM16F. Specifically, proteoliposomes containing TMEM16F prepared with n-dodecyl-β-D-maltoside (DDM) exhibited faster scrambling kinetics and higher Ca2+ sensitivity than those prepared with other detergents. Unexpectedly, blue native gel electrophoresis revealed that the DDM-isolated TMEM16F contained additional higher-order assemblies, which was further supported by size-exclusion chromatography and atomic force microscopy. Collectively, these findings suggest that the preparation process with different detergents influences the oligomeric states and scramblase activities of TMEM16F. Our results suggest a potential link between the structural plasticity and function of TMEM16F and provide a critical framework for detergent choice in structural and functional studies of membrane protein complexes.