Studies of Protein Phase Separation Using Leishmania Kinetoplastid Membrane Protein-11.

Despite the significant understanding of phase separation in proteins with intrinsically disordered regions, a considerable percentage of proteins without such regions also undergo phase separation, presenting an intriguing area for ongoing research across all kingdoms of life. Using a combination of spectroscopic and microscopic techniques, we report here for the first time that a low temperature and low pH can trigger the liquid-liquid phase separation (LLPS) of a parasitic protein, kinetoplastid membrane protein-11 (KMP-11). Electrostatic and hydrophobic forces are found to be essential for the formation and stability of phase-separated protein assemblies. We show further that the increase in the ionic strength beyond a threshold decreases the interchain electrostatic interactions acting between the alternate charged blocks, altering the propensity for phase separation. More interestingly, the addition of cholesterol inhibits LLPS by engaging the cholesterol recognition amino acid consensus (CRAC)-like domains present in the protein. This was further confirmed using a CRAC-deleted mutant with perturbed cholesterol binding, which did not undergo LLPS.