Liquid-liquid phase separation in the formation of enzymatic microreactors

• Grant number: 23/12987-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2023
• End date: October 31, 2024
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Antonio José da Costa Filho
• Grantee: João Paulo de Mauro Faccin
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Liquid-liquid phase separation (LLPS) is a well-known phenomenon in areas such as polymer physical chemistry, materials science, and engineering. In LLPS, a thermodynamically favorable phase separation gives rise to liquid droplets rich in a solute (dense phase) that become dispersed in a dilute liquid phase. In Cell Biology, LLPS has received recent attention after the demonstration of its existence and relevance for the compartmentalization of the intracellular space. In a derivation of the cellular findings, LLPS involving biological molecules such as proteins and nucleic acids have been explored in biotechnological applications such as drug delivery, biosensors, and microreactor production. The present proposal fits into this last application and consists of producing a chimera protein containing a globular protein with potential biotechnological interest (and that would not be able to undergo LLPS) conjugated to domains of low complexity (LCDs). In this situation, LCDs are used as molecular adhesives to promote the LLPS of the chimera protein, thus giving rise to a biomolecular condensate rich in the protein of interest. This technique can be used in many applications, such as water treatment, tissue engineering, vaccine development, biosensors, and food engineering. In this project, the enzyme cytochrome P450 is the protein of interest. Its participation in numerous biological processes, such as the production of antibiotics, the metabolism of drugs in mammalian cells, and the detoxification of xenobiotics, justifies this interest. The P450 will be combined with sequences of LCDs already reported in the literature. Once this is done, the characterization of the condensate in terms of its physicochemical properties and enzymatic activity will be carried out. To characterize the separated phases formed, there will be structural analyses of the chimera protein formed through differential scanning calorimetry (DSC), circular dichroism (CD), and fluorescence. For the observation of the phases, the techniques of microscopy and dynamic light scattering (DLS) will be used. We hope to be able to consolidate the method in our research group through the production of the chimera P450-LCD, thus extending our expertise and finding room for new applications.