DEVELOPMENT AND USE OF FLUORESCENCE TECHNIQUES FOR THE STUDY OF PROTEIN CONFORMATIONAL CHANGES

• Grant number: 11/20941-9
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: April 01, 2012
• End date: July 31, 2016
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Grantee: Marcelo Ferreira Marcondes Machado
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 12/50191-4 - Synthesis, kinetic studies and applications of substrates and inhibitors for proteolytic enzymes, AP.TEM
• Associated scholarship(s): 14/00661-0 - Development and use of fluorescence techniques for the study of protein conformational changes, BE.EP.PD

Abstract

The main goal of the present project is to study the hinge movement that the peptidase, timet oligopeptidase (TOP) may undergo upon substrate or inhibitor binding. Fluorescence techniques will be used to study this conformational change in great detail. TOP constructs will be prepared equipped with two donor-donor fluorescent probes (Trp residues) or donor-acceptor probes (5-F Trp - p-nitro-phenylalanine) pairs, one at each side of the central deep channel of the TOP structure.Fluorescence experiments, in steady state and time-resolved mode will inform us about the hinge movement in this enzyme and the impact of different substrates and inhibitors on it. The kinetics of the hinge movement will be investigated using the same construct via stopped-flow fluorescence spectroscopy. A better understanding of the hinge movement will allow us to tune the activity of this enzyme and provides a good basis for designing new efficient inhibitors. We foresee that once we successful demonstrate the potential of this approach especially the 5-FTrp - p-nitro-phenylalanine donor-acceptor pair as outlined in this application, this approach will be adopted by other researchers aiming to study protein conformational changes.

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)

MARCONDES, M. F. M.; ALVES, F. M.; ASSIS, D. M.; HIRATA, I. Y.; JULIANO, L.; OLIVEIRA, V.; JULIANO, M. A.. Substrate specificity of mitochondrial intermediate peptidase analysed by a support-bound peptide library. FEBS OPEN BIO, v. 5, p. 429-436, 2015. (12/50191-4, 11/20941-9, 14/00661-0)

GOMES SMAUL, MAYRIM MACHADO; BUDU, ALEXANDRE; MONTAGNA, GEORGINA NURI; DA SILVA FERRARA, TAISE FERNANDA; MALUF, SARAH EL CHAMY; BAGNARESI, PIERO; FERREIRA MACHADO, MARCELO MARCONDES; DOS SANTOS, FELLIPE BRONZE; DE AZEVEDO, MAURO FERREIRA; CARMONA, ADRIANA KARAOGLANOVIC; et al. Plasmodium falciparum histidine triad protein and calmodulin modulates calcium homeostasis and intracellular proteolysis. Biochemical and Biophysical Research Communications, v. 503, n. 2, p. 722-728, SEP 5 2018. (12/24278-5, 16/15298-3, 14/07138-0, 16/02412-2, 15/19316-3, 13/12913-0, 15/00689-4, 15/11861-2, 15/07182-2, 11/20941-9)

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. Probing the Conformational States of Thimet Oligopeptidase in Solution. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, v. 23, n. 13, p. 18-pg., 2022-07-01. (14/00661-0, 11/20941-9, 20/09678-3, 18/09158-0, 11/51989-7)

OLIVEIRA-SOUZA, WELLINGTON P.; BRONZE, FELLIPE; BROOS, JAAP; MARCONDES, MARCELO F. M.; OLIVEIRA, VITOR. On the efficient bio-incorporation of 5-hydroxy-tryptophan in recombinant proteins expressed in Escherichia coli with T7 RNA polymerase-based vectors. Biochemical and Biophysical Research Communications, v. 492, n. 3, p. 343-348, OCT 21 2017. (14/20847-0, 11/20941-9, 14/00661-0)