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Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence.


ABSTRACT: Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6?kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635?nm and fluoresces at 670?nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625?nm and fluoresces at 657?nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.

SUBMITTER: Rumyantsev KA 

PROVIDER: S-EPMC4683375 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence.

Rumyantsev Konstantin A KA   Shcherbakova Daria M DM   Zakharova Natalia I NI   Emelyanov Alexander V AV   Turoverov Konstantin K KK   Verkhusha Vladislav V VV  

Scientific reports 20151218


Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, r  ...[more]

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