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. Author manuscript; available in PMC: 2013 Oct 24.
Published in final edited form as: J Am Chem Soc. 2012 Oct 10;134(42):17582–17591. doi: 10.1021/ja3064292

Figure 1.

Figure 1

Immunoprobed isoelectric focusing allows dynamic and immunoreactivity-verified monitoring of GFP isoform dynamics during reversible photobleaching. (A) Reversible photobleaching upon UV illumination creates dark GFP isoforms with increased pIs relative to their bright “parents”. (B) Microfluidic chip with three parallel channels between each pair of access wells. Dynamic isoelectric photoswitching processes can be monitored in real time, or isoforms can be captured by the LAVAgel matrix and probed in situ with fluorescently labeled anti-GFP antibody. (C) A sketch of avGFP chromophore and proton wire dynamics29, 40, 41, 56. Hydrogen bond networks allow proton exchange of the chromophore pocket with the external solvent. Glu222 is involved in excited state proton transfer (ESPT) with the chromophore Tyr66. The E222G mutation in acGFP inhibits ESPT and perhaps proton exchange with the internal wire proposed by Agmon et al.41, 55. The dependence of reversible photobleaching magnitude on pH suggests involvement of a titratable residue, X, in the chromophore vicinity that affects chromophore protonation state by hydrogen bonding38 (see text for details).