AsianScientist (Nov. 7, 2011) – A team of researchers from China has developed a fusion protein that may be used as an intracellular sensor for reduced nicotinamide adenine dinucleotide (NADH), an essential cofactor in cells.
The genetically encoded protein combines the Rex protein from bacteria and a genetic mutant protein called circularly permuted YFP (cpYFP) from jellyfish.
NADH and its oxidized form, NAD+, are important coenzymes inside cells involved in many cellular activities such as energy metabolism, mitochondrial function, carcinogenesis, biosynthesis, and gene expression.
When NADH is present and binds to Rex, the conformational change of Rex activates YFP to generate fluorescence. The new technique is useful as it allows for dynamic monitoring of NADH inside the cell both temporally and spatially.
Existing methods to probe the concentration of NADH in vitro are limited, and fail to distinguish NADH from NADPH, a similar molecule with distinct functions.
In nature however, organisms from bacteria to mammalians have proteins that are evolved to directly sense the intracellular concentration of NADH, and among them, the Rex protein is reported to be the most sensitive.
In this study, the researchers combined the Rex protein with cpYFP, a circularly permuted fluorescence protein which is highly sensitive to its microenvironment and widely used in protein engineering to develop biosensors.
Using the fusion protein as a fluorescence sensor, they found that upon perturbation in mammalian cells, cytosolic NADH is more sensitive to environmental changes while mitochondria have a strong tendency to maintain homeostasis, or constant NADH levels.
These new sensors provide an alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor.
The article can be found at: Zhao Y et al. (2011) Genetically Encoded Fluorescent Sensors for Intracellular NADH Detection.
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Source: Cell Press.
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