Design and characterization of micro-reactors constituted by the protein chlorocatechol 1,2-dioxygenase and low complexity domains

• Grant number: 20/15542-7
• Support Opportunities: Regular Research Grants
• Start date: July 01, 2021
• End date: December 31, 2023
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Antonio José da Costa Filho
• Grantee: Antonio José da Costa Filho
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated researchers: Ana Paula Ulian de Araujo ; Eduardo Festozo Vicente
• Associated scholarship(s): 22/07049-4 - Expression, purification, and characterization of chimera proteins, BP.TT; 21/08956-2 - Expression, purification, and characterization of chimera enzymes, BP.TT

Abstract

Low complexity domains (LCDs) are sequences of amino acids with very peculiar structural features and still poorly explored in Biochemistry and Molecular Biophysics. Their dynamics of interactions as well as their functions, toxicity and persistence during evolution still lack better understanding. It is known, tough, that if used as molecular adhesives in proteins (so called- chimeras), the LCDs are capable of promoting phase separation (agglomerates), such as in droplets (liquid-liquid phase separation) or in solid phase (irreversible aggregates). These phases formed by chimeras can have their chemical surfaces controlled so as to maintain the original physical/chemical properties of the protein. This strategy allows for a great number of applications, such as tissue and food engineering, vaccine development, biosensors, among others. In this grant proposal, we intend to use the enzyme chlorocatechol 1,2-dioxygenase (1,2-CCD), which degrades polycyclic aromatic compounds, and, therefore, has potential use in bioremediation, as a case study to build enzymatic micro-reactors formed by that enzyme and LCDs. As a control, we will produce micro-reactors formed by the Green Fluorescent Protein and LCDs, a system already reported as forming separate liquid phases. The enzyme will be expressed and purified with LCDs bound to its N- and C-terminals. The LCDs will be derived from low complexity sequences already reported in the literature to undergo liquid-liquid phase separation. To characterize the chimera proteins, we will use differential scanning calorimetry (DSC), circular dichroism (CD) and measurements of the enzymatic activity. To monitor the phase separation, we will use microscopic techniques and light scattering. We expect the phase separated aggregates show potential to be used in degrading aromatic compounds and the know how gained by our group to be extended to other enzymes in biotechnological application. (AU)

Scientific publications

(4)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

MENDES, LUIS FELIPE S.; COSTA-FILHO, ANTONIO J.. A gold revision of the Golgi Dynamics (GOLD) domain structure and associated cell functionalities. FEBS Letters, v. 596, n. 8, p. 18-pg., 2022-02-14. (17/24669-8, 20/15542-7, 15/50366-7, 21/10465-7)

ALVAREZ, NATALIA; FREDDI, PRISCILLA; CASTELLANI, STEPHANIE; VEIGA, NICOLAS; FACCHIN, GIANELLA; COSTA-FILHO, ANTONIO J.. New Insights into the Biophysical Behavior of an Old Molecule: Experimental and Theoretical Studies of the Interaction Between 1,10-Phenanthroline and Model Phospholipid Membranes. Brazilian Journal of Physics, v. 52, n. 4, p. 8-pg., 2022-08-01. (15/50366-7, 20/15542-7)

MENDES, LUIS FELIPE S.; GIMENES, CAROLINA O.; DA SILVA, MARILIA D. O.; ROUT, SAROJ K.; RIEK, ROLAND; COSTA-FILHO, ANTONIO J.. The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion. Protein Science, v. 33, n. 7, p. 16-pg., 2024-07-01. (22/06006-0, 15/50366-7, 09/54044-3, 21/10465-7, 20/15542-7)

EVANGELISTA, NATHAN N.; MICHELETTO, MARIANA C.; KAVA, EMANUEL; MENDES, LUIS F. S.; COSTA-FILHO, ANTONIO J.. Biomolecular condensates of Chlorocatechol 1,2-Dioxygenase as prototypes of enzymatic microreactors for the degradation of polycyclic aromatic hydrocarbons. International Journal of Biological Macromolecules, v. 270, p. 9-pg., 2024-05-15. (17/24669-8, 20/01152-2, 18/13016-6, 15/50366-7, 21/10795-7, 20/15542-7)