The structure of the chromophore within DsRed, a red fluorescent protein from coral
The structure of the chromophore within DsRed, a red fluorescent protein from coral
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DOI：
10.1073/pnas.97.22.11990
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发表时间：
2000-10-24
影响因子：
11.1
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Gross, LA;Baird, GS;Tsien, RY
DsRed是一种明亮的红色荧光蛋白，最近从Discosoma珊瑚中克隆，与水母Aequorea的绿色荧光蛋白（GFP）同源。DsRed生物化学中的一个核心问题是未成熟DsRed的GFP样475-nm激发和500-nm发射最大值红移到成熟DsRed的558-nm激发和583-nm发射最大值的机制，用赖氨酰内肽酶消化成熟的DsRed后，纯化的带有发色团的肽的高分辨率质谱显示，相对于从保持绿色的突变体K83 R类似地制备的肽，一些分子损失了2 Da。串联质谱法表明Gln-66的α-碳和氮之间的键在DsRed中已经脱氢，通过在咪唑啉酮的2-位形成-C=N-C=O来延伸GFP发色团。根据量子力学计算，该酰亚胺取代基定量地解释了红移。酰基亚胺中C=N键的可逆水合作用可以解释为什么变性会将成熟的DsRed移回GFP样吸光度。C=N键在沸腾时水解，解释了为什么DsRed在SDS/PAGE上显示两个片段条带。该测定表明，即使在长时间老化后，从绿色到红色发色团的转化仍然不完全。
DsRed, a-brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea, A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed, After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming -C=N-C=O at the 2-position of the imidazolidinone, This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C=N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C=N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.