1jc0
CRYSTAL STRUCTURE ANALYSIS OF A REDOX-SENSITIVE GREEN FLUORESCENT PROTEIN VARIANT IN A REDUCED FORM
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OverviewOverview
Current methods for determining ambient redox potential in cells are, labor-intensive and generally require destruction of tissue. This, precludes single cell or real time studies of changes in redox poise that, result from metabolic processes or environmental influences. By, substitution of surface-exposed residues on the Aequorea victoria green, fluorescent protein (GFP) with cysteines in appropriate positions to form, disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been, created. roGFPs have two fluorescence excitation maxima at about 400 and, 490 nm and display rapid and reversible ratiometric changes in, fluorescence in response to changes in ambient redox potential in vitro, and in vivo. Crystal structure analyses of reduced and oxidized crystals, of roGFP2 at 2.0- and 1.9-A resolution, respectively, reveal in the, oxidized state a highly strained disulfide and localized main chain, structural changes that presumably account for the state-dependent, spectral changes. roGFP1 has been targeted to the mitochondria in HeLa, cells. Fluorometric measurements on these cells using a fluorescence, microscope or in cell suspension using a fluorometer reveal that the, roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status, and responds to membrane-permeable reductants and oxidants. The roGFP1, probe reports that the matrix space in HeLa cell mitochondria is highly, reducing, with a midpoint potential near -360 mV (assuming mitochondrial, pH approximately 8.0 at 37 degrees C). In other work (C. T. Dooley, T. M., Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is, also unusually reducing but somewhat less so than the mitochondrial, matrix.
About this StructureAbout this Structure
1JC0 is a Single protein structure of sequence from Aequorea victoria. Full crystallographic information is available from OCA.
ReferenceReference
Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators., Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ, J Biol Chem. 2004 Mar 26;279(13):13044-53. Epub 2004 Jan 13. PMID:14722062
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