Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics.
Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics.
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DOI：
10.1038/srep37362
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发表时间：
2016-11-18
影响因子：
4.6
通讯作者：
Kennis JT
中科院分区：
文献类型：
--
作者：
Hontani Y;Shcherbakova DM;Baloban M;Zhu J;Verkhusha VV;Kennis JT
由细菌光敏色素（BphPs）工程化的近红外荧光蛋白（NIR FP）对于体内成像具有极大的兴趣。它们利用胆绿素（BV）作为发色团，这是一种血红素降解产物，因此它们直接用于哺乳动物组织。在这里，我们报告的荧光特性的近红外FP的关键改变，在其BV结合位点。BphP 1-FP、iRFP 670和iRFP 682在PAS和GAF结构域中都具有Cys残基，而不是像野生型BphP那样仅在PAS结构域中具有Cys残基。我们发现在GAF中具有Cys或在PAS和GAF两者中具有Cys的NIR FP变体显示具有长荧光寿命的蓝移发射。相比之下，突变体中的PAS只有半胱氨酸或没有半胱氨酸残基在所有表现出红移发射寿命较短。结合这些结果与以前的生物化学和BphP 1-FP结构数据，我们得出结论，BV加合物结合到Cys的GAF是明亮的蓝移荧光的起源。我们提出，长荧光寿命来自（i）空间上更受约束的硫醚键，使环A的迁移率低于典型的BphPs，以及（ii）π-电子共轭不在环A上延伸，使激发态失活对环A迁移率不太敏感。
Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.
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影响因子：
2.4
作者：
Tran MT;Tanaka J;Hamada M;Sugiyama Y;Sakaguchi S;Nakamura M;Takahashi S;Miwa Y
通讯作者：
Miwa Y
影响因子：
4.3
作者：
Marx, Ailie;Adir, Noam
通讯作者：
Adir, Noam
DOI：
10.1073/pnas.0407645101
发表时间：
2004-12-14
影响因子：
11.1
作者：
Fischer, AJ;Lagarias, JC
通讯作者：
Lagarias, JC
DOI：
10.1107/s0907444912020537
发表时间：
2012-08-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Bellini, Dom;Papiz, Miroslav Z.
通讯作者：
Papiz, Miroslav Z.
影响因子：
4.1
作者：
Karniol, B;Wagner, JR;Vierstra, RD
通讯作者：
Vierstra, RD