Polypeptides in Order to Illuminate Redox States

„Attachment of linkers between a special pair of green fluorescent proteins (GFP) shows great promise for developing genetically encoded redox sensitive biosensors,“ said Vladimir L. Kolossov from the University of Illinois‘ Institute for Genomic Biology (IGB). To detect oxidation and reduction, the biosensor uses the optical technique Fluorescence resonance energy transfer (FRET).

Photo: Principle of redox-biosensor
Process on the cellular level; © Society for Experimental Biology and Medicine

The absence of polypeptide linkers able to sense the redox state by undergoing a conformational change was the major obstacle to a FRET-based redox sensor. The researchers designed the linker sequence such that in its reduced state the linker is an α-helix. Thiol groups, strategically placed throughout the linker, sense the redox potential of the environment and form disulfide bonds upon oxidation.

Under oxidative conditions intramolecular disulfide bonds can form, shifting the free energy minimum from the α-helix, to a „clamped-coil“ state (similar to a helix-coil transition). The coiled state allows the two fluorescent proteins to approach closer than in the extended helix state, where they can more efficiently exchange excitation energy which means a high FRET state. The extent of energy transfer is easily quantified from the increased emission of the acceptor.

This is the first step towards development of a FRET-based biosensor for visualizing redox potentials and oxidative stress in live cells and tissues via optical microscopy.

„We employed a sensitive technique for measuring FRET to screen our linkers. This methodology greatly expedited the quantitative analysis and development of the linkers and will be very useful for the development of other FRET-based sensors,“ said Bryan Q. Spring, a doctoral student. Given the importance of the intracellular redox state in determining a cell‘s fate, FRET-based redox sensors offer promise for understanding molecular mechanisms underlying human health and disease.

COMPAMED.de; Source: Society for Experimental Biology and Medicine