Construction and characterization of micro reactors of chlorocatechol 1,2-dioxygenase that utilize low complexity domains as molecular adhesives

• Grant number: 20/01152-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): August 01, 2020
• Effective date (End): June 30, 2022
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Antonio José da Costa Filho
• Grantee: Nathan Nunes Evangelista
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Low complexity domains (LCDs) are aminoacid sequences with peculiar structural features that are still poorly exploited in biophysics and biochemistry. The dynamics of their interactions is not known as well as their functions, toxicity and persistence throughout evolution. It is know, however, that if used as molecular adhesives in proteins (so-called chimera proteins), the LCDs are capable of promoting phase separation, such as in droplets (liquid-liquid separation) or in solids (irreversible aggregates). Those phases can thus have their surface properties controlled, while maintaining the physo-chemical properties of the original protein. This strategy allows for a variety of applications, such as in water treatment, tissue engineering, vaccine development, biosensors, among others. In this project, we will use the enzyme chlorocatechol 1,2-dioxygenase (1,2-CCD), which plays a role in the degradation of polycyclic aromatic compounds with potential use in bioremediation schemes. The enzyme will be expressed and purified with LCDs attached to its N- and C-terminals. The LCDs used will come from previously reported sequences that showed capacity to induce liquid-liquid phase separation. To characterize the chimera protein, we will perform structural assays via differential scanning calorimetry (DSC), circular dichroism, (CD) and enzyme activity measurements. To direct observe phase formation, we will use microscopic methods and dynamics light scattering (DLS). We expect the final phase separated aggregate of the chimera protein to be useful as a micro reactor for degradation of aromatic compounds.