TY - JOUR
T1 - Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence
AU - Rumyantsev, Konstantin A.
AU - Shcherbakova, Daria M.
AU - Zakharova, Natalia I.
AU - Emelyanov, Alexander V.
AU - Turoverov, Konstantin K.
AU - Verkhusha, Vladislav V.
N1 - Publisher Copyright:
© 2015, Nature Publishing Group. All rights reserved.
PY - 2015/12/18
Y1 - 2015/12/18
N2 - 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 19.6kDa, 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 635nm and fluoresces at 670nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625nm and fluoresces at 657nm. 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.
AB - 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 19.6kDa, 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 635nm and fluoresces at 670nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625nm and fluoresces at 657nm. 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.
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U2 - 10.1038/srep18348
DO - 10.1038/srep18348
M3 - Article
C2 - 26679720
AN - SCOPUS:84951188493
SN - 2045-2322
VL - 5
JO - Scientific reports
JF - Scientific reports
M1 - 18348
ER -