Martynov V I, Savitsky A P, Martynova N Y, Savitsky P A, Lukyanov K A, Lukyanov S A
Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia.
J Biol Chem. 2001 Jun 15;276(24):21012-6. doi: 10.1074/jbc.M100500200. Epub 2001 Mar 19.
Anemonia sulcata purple protein (asFP595) belongs to a family of green fluorescent protein (GFP)-like proteins from the Anthozoa species. Similar to GFP, asFP595 apparently forms its chromophore by modifying amino acids within its polypeptide chain. Until now, the GFP-like proteins from Anthozoa were thought to contain chromophores with the same imidazolidinone core as GFP. Mass spectral analysis of a chromophore-containing tryptic pentapeptide from asFP595 demonstrates that chromophore formation in asFP595 is stoichiometrically the same as that in GFP: one H(2)O and two H(+) are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzylidene)-6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals three pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maximum = 430 nm) at pH 3.0; red (absorption maximum = 535 nm) at pH 8.0; and colorless (absorption maximum = 380 nm) at pH 14.0. The pK(a) values for these spectral transitions (6.8 and 10.9) are consistent with the ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP. When the asFP595 chromophore cyclizes, the Cys-Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 shows that a pentapeptide with the cleaved Cys-Met bond is the only fragment associated with the red-shifted absorbance. These results imply that fragmentation of asFP595 is a critical step in protein maturation.
沟迎风海葵紫色蛋白（asFP595）属于来自珊瑚纲物种的一类绿色荧光蛋白（GFP）样蛋白。与GFP相似，asFP595显然是通过修饰其多肽链内的氨基酸来形成发色团的。到目前为止，人们认为来自珊瑚纲的GFP样蛋白含有与GFP具有相同咪唑烷酮核心的发色团。对来自asFP595的含发色团的胰蛋白酶解五肽进行质谱分析表明，asFP595中发色团的形成在化学计量上与GFP相同：释放一分子H₂O和两分子H⁺，同时形成一个席夫碱和脱氢酪氨酸。然而，对该asFP595发色肽的结构研究表明，与GFP不同，发色团环化需要另一个肽键氮和羰基碳，该反应产生六元杂环2-(4-羟基亚苄基)-6-羟基-2,5-二氢吡嗪。分光光度滴定揭示了asFP595发色肽的三种pH依赖性形式：pH 3.0时为黄色（最大吸收波长 = 430 nm）；pH 8.0时为红色（最大吸收波长 = 535 nm）；pH 14.0时为无色（最大吸收波长 = 380 nm）。这些光谱转变的pKₐ值（6.8和10.9）分别与脱氢酪氨酸酚羟基的电离和发色团杂环中脒阳离子的去质子化一致。asFP595中的脒基团导致了该蛋白独特的吸收光谱，相对于GFP的吸收光谱有显著的红移。当asFP595发色团环化时，与发色团相邻的半胱氨酸-甲硫氨酸键水解，将发色蛋白裂解为8 kDa和20 kDa的片段。对变性asFP595的胰蛋白酶消化产物进行高效液相色谱分析表明，具有裂解的半胱氨酸-甲硫氨酸键的五肽是唯一与红移吸光度相关的片段。这些结果表明asFP595的裂解是蛋白质成熟的关键步骤。
