Structural basis for bathochromic shift of fluorescence in far-red fluorescent proteins eqFP650 and eqFP670
Structural basis for bathochromic shift of fluorescence in far-red fluorescent proteins eqFP650 and eqFP670
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DOI：
10.1107/s0907444912020598
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发表时间：
2012-09-01
期刊：
影响因子：
--
通讯作者：
Pletnev, Vladimir
中科院分区：
文献类型：
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作者：
Pletnev, Sergei;Pletneva, Nadya V.;Pletnev, Vladimir
远红荧光蛋白（FPs） eqFP650 (lambda(max)(ex)/lambda（max)(em) 592/650 nm）和eqFP670 (lambda(max)(ex)/lambda（max)(em) 605/670 nm）分别在1.8和1.6埃分辨率下测定了远红荧光蛋白Katushka (lambda(max)(ex)/lambda（max)(em) 588/635 nm）的晶体结构。对结构的检查表明，有两组变化负责这些蛋白质的激发/发射带相对于它们的前身的变色。由于eqFP650和eqFP670中分别存在一个和三个水分子，第一组变化导致发色团的酰基胺位点亲水性增加。这些水分子通过氢键将发色团与蛋白质支架连接起来，导致eqFP650和eqFP670发射带发生类似于15 nm的色移。在eqFP670中观察到的第二组变化源于天冬酰胺取代了143和158号丝氨酸。eqFP670的Asn143和Asn158彼此之间，以及与蛋白质支架和与发色团的对羟基成氢键，导致eqFP670的发射带比eqFP650增加了类似20 nm的色移。观察到的结构变化的作用被诱变证实。
The crystal structures of the far-red fluorescent proteins (FPs) eqFP650 (lambda(max)(ex)/lambda(max)(em) 592/650 nm) and eqFP670 (lambda(max)(ex)/lambda(max)(em) 605/670 nm), the successors of the far-red FP Katushka (lambda(max)(ex)/lambda(max)(em) 588/635 nm), have been determined at 1.8 and 1.6 angstrom resolution, respectively. An examination of the structures demonstrated that there are two groups of changes responsible for the bathochromic shift of excitation/emission bands of these proteins relative to their predecessor. The first group of changes resulted in an increase of hydrophilicity at the acylimine site of the chromophore due to the presence of one and three water molecules in eqFP650 and eqFP670, respectively. These water molecules provide connection of the chromophore with the protein scaffold via hydrogen bonds causing an similar to 15 nm bathochromic shift of the eqFP650 and eqFP670 emission bands. The second group of changes observed in eqFP670 arises from substitution of both Ser143 and Ser158 by asparagines. Asn143 and Asn158 of eqFP670 are hydrogen bonded with each other, as well as with the protein scaffold and with the p-hydroxyphenyl group of the chromophore, resulting in an additional similar to 20 nm bathochromic shift of the eqFP670 emission band as compared to eqFP650. The role of the observed structural changes was verified by mutagenesis.