2014
DOI: 10.1002/cbic.201402075
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Replacement of Highly Conserved E222 by the Photostable Non‐photoconvertible Histidine in GFP

Abstract: The widely used green fluorescent protein (GFP) decarboxylates upon irradiation; this involves removal of the acidic function of the glutamic acid at position 222, thereby resulting in the irreversible photoconversion of GFP. To suppress this phenomenon, the photostable, non-photoconvertible histidine was introduced at position 222 in GFP. The variant E222H shows negligible photodynamic processes and high expression yield. In addition, the stable and bright fluorescence over a wide pH range makes the E222H pro… Show more

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Cited by 6 publications
(4 citation statements)
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“…Previous studies in Bio-HLEDs have focused on classical FPs like enhanced GFP (eGFP), mCherry, and R-phycoerythrin, which are not well suited due to their propensity to exhibit photobleaching under irradiation, pH, and thermal stress scenarios. In light of these limitations, we have resorted to design an eGFP variant (eGFP-AA) implementing nine point-mutations in the structure of eGFP merging those previously used in the eGFP mutants eGFP-E222H 51 and eGFP-FF 52 (Fig. 1 and Supplementary Figs.…”
Section: Fluorescent Protein Designmentioning
confidence: 99%
“…Previous studies in Bio-HLEDs have focused on classical FPs like enhanced GFP (eGFP), mCherry, and R-phycoerythrin, which are not well suited due to their propensity to exhibit photobleaching under irradiation, pH, and thermal stress scenarios. In light of these limitations, we have resorted to design an eGFP variant (eGFP-AA) implementing nine point-mutations in the structure of eGFP merging those previously used in the eGFP mutants eGFP-E222H 51 and eGFP-FF 52 (Fig. 1 and Supplementary Figs.…”
Section: Fluorescent Protein Designmentioning
confidence: 99%
“…4B). Among the isosteric residues, 52 histidine represents a suitable candidate because of its H-bond-donating capability. Through site-directed mutagenesis, the resultant single mutant E222H exhibits the remarkably improved brightness with FQY = 0.425, comparable to other bright RFPs.…”
Section: Resultsmentioning
confidence: 99%
“…While there are examples of structured His-tags appearing in the electron density for structures in the PDB, we have not been able to find any previous case where it is incorporated into the fused protein as a functional element. Previous studies have been conducted in which E 222 on strand 11 18 and T 203 on strand 10 19 have been mutated to histidine. The absorbance spectra of these mutants are qualitatively similar to those of H6:GFP:loop:s11 10 and H6:GFP:loop:s10 (Figure S2).…”
Section: ■ Results and Discussionmentioning
confidence: 99%