Design of a GFP-based non invasive biosensor to NADP+(H) redox state in living cells

MOLINARI, P.E; ZURBRIGGEN, M.; BUSTOS-SANMAMED, P; A R KRAPP, CARRILLO, N.

Córdoba

Congreso; LII Reunión de SAIB; 2016

SAIB

One key component of central metabolism in all life forms is NADP+(H). Current methods for NADP+(H) determination in biological samples are hampered by lack of sensitivity and reproducibility, precluding studies of redox changes in living cells. The recent development of redox-sensitive green fluorescent proteins (roGFPs) as genetically encoded probes allowed spatiotemporal detection of thiol-containing redox metabolites in vivo. We designed a biosensor to determine the cellular redox state of NADP+(H), by fusing roGFP to the coding region of rice NADP+(H) thioredoxin reductase C (NTRC) via spacer arms of variable lengths (30 and 45 residues). NTRC allows transduction of redox signals from the world of pyridine nucleotides to that of thiol-disulfides. The NTRC-roGFP fusions display reversible ratiometric fluorescence changes in response to the redox potential of NADP+(H). In vitro characterization of both fusion proteins indicated that ratiometric values varied with the redox potential of NADP+(H) as predicted by the Nernst equation, permitting its accurate determination by measuring the oxidation state of the probe. As a proof-of-concept test we transformed Escherichia coli cells and observed fluorescence changes by confocal microscopy in response to the redox state of the organism. This biosensor can be used to study the NADP+(H) redox poise of cells and organelles in real time.