| Full text | |
| Author(s): |
Marcondes, Marcelo F. M.
;
Santos, Gabriel S.
;
Bronze, Fellipe
;
Machado, Mauricio F. M.
;
Perez, Katia R.
;
Hesselink, Renske
;
de Vries, Marcel P.
;
Broos, Jaap
;
Oliveira, Vitor
Total Authors: 9
|
| Document type: | Journal article |
| Source: | INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES; v. 23, n. 13, p. 18-pg., 2022-07-01. |
| Abstract | |
Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2). (AU) | |
| FAPESP's process: | 14/00661-0 - Development and use of fluorescence techniques for the study of protein conformational changes |
| Grantee: | Marcelo Ferreira Marcondes Machado |
| Support Opportunities: | Scholarships abroad - Research Internship - Post-doctor |
| FAPESP's process: | 11/20941-9 - DEVELOPMENT AND USE OF FLUORESCENCE TECHNIQUES FOR THE STUDY OF PROTEIN CONFORMATIONAL CHANGES |
| Grantee: | Marcelo Ferreira Marcondes Machado |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |
| FAPESP's process: | 20/09678-3 - Selection of inhibitors for the NS2/NS3 proteases from Dengue and Zika viruses by cellular high-throughput assays coupled with cyclic peptide libraries |
| Grantee: | Vitor Marcelo Silveira Bueno Brandão de Oliveira |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 18/09158-0 - Prolyl oligopeptidase: structure-activity and secretion |
| Grantee: | Vitor Marcelo Silveira Bueno Brandão de Oliveira |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 11/51989-7 - Jaap Broos | University of Groningen - Holanda |
| Grantee: | Vitor Marcelo Silveira Bueno Brandão de Oliveira |
| Support Opportunities: | Research Grants - Visiting Researcher Grant - International |