Membraneless Organelles by Design: The Carboxysome
SESSION 5: PEPTIDE MATERIALS AND DELIVERY
Monday, June 26, 2023, at 11:35 am - 11:55 am
Membraneless organelles are widespread in biology, and their role in supporting cellular function is increasingly recognized. They form by liquid-liquid phase separation, LLPS, or coacervation of proteins and/or protein-nucleic acid mixtures, for example, by complex coacervation or associative LLPS of a positively charged macromolecule, for example, histone proteins, and a negatively charged macromolecule, for example, DNA, via electrostatic interactions. This mechanism leads to compartmentalization of the components within liquid droplets that can sequester solutes ranging from small molecules to proteins by partitioning, leading to dynamic control of function such as enzyme and ribozyme activity.
Here, we develop a bottom-up approach for creating functional designed organelles using complex coacervation between proteins with charged surfaces and a counter charged polypeptide. We started with a negative supercharged mutant of apo cytochrome b562: the electrostatic repulsion due to close packing of the surface charges leads to the destabilization of the molten globule state resulting in an intrinsically disordered protein, IDP. Folding is rescued by counterions - for example by high concentrations of NaCl or low concentration of CaCl2; binding to heme further restores a native-like structure.
We previously showed that cyt b562 catalyzes hydrogen production and carbon dioxide reduction when the native heme is replaced with cobalt protoporphyrin IX, CoPPIX. Supercharged CoPPIX-cyt b562 conserves folding and function of the artificial enzyme in the presence of salts. Mixing with poly-Arg results in complex coacervation as observed by turbidimetric assays and optical microscopy. The droplets contain folded CoPPIX-cyt b562(-22) that display circular dichroism and UV-vis signatures identical to WT. The droplets catalyse production of molecular hydrogen and reduction of carbon dioxide with efficiency higher than WT. Surprisingly, the photosensitizer used in the reaction, Ru(bpy)3, partitions spontaneously into the droplets and may contribute to the observed increase in activity.
Our results show that the supercharging of a protein creates a viable avenue for complex coacervation with an oppositely charged polymer, without interfering with function. Our ultimate goal is to create a functional membraneless organelle analogous to a carboxysome.
Giovanna Ghirlanda is a professor of chemistry at Arizona State University. Her research interests are in chemical biology, protein chemistry, and protein engineering. Professor Ghirlanda received a BSc/MSc degree in Medicinal Chemistry and a Ph.D. in Organic Chemistry from the University of Padova, Italy, with a thesis on the redox catalytic properties of supramolecular metallocomplexes.
Prior to joining ASU, Giovanna pursued postdoctoral work at the University of Pennsylvania with Professor William F. DeGrado, now at the University of California at San Francisco, specializing on de novo design of peptides and proteins. Her research is in the area of chemical biology and focuses on the design of functional proteins. A major thrust is in the area of sustainable fuel production, designing protein-based hybrid metalloenzymes that catalyze hydrogen production and carbon dioxide reduction in mild conditions. Other projects in the lab include the design of antiviral lectins and work on protein folding in WW domains, in collaboration with Professor Banu Ozkan, Physics, ASU.