PDF(Pubmed)
Abstract：
Cyanobacteriochrome (CBCR)-derived fluorescent proteins are a class of reporters that can bind bilin cofactors and fluoresce across the ultraviolet to the near-infrared spectrum. Derived from phytochrome-related photoreceptor proteins in cyanobacteria, many of these proteins use a single small GAF domain to autocatalytically bind a bilin and fluoresce. The second GAF domain of All1280 (All1280g2) from Nostoc sp. PCC7120 is a DXCF motif-containing protein that exhibits blue-light-responsive photochemistry when bound to its native cofactor, phycocyanobilin. All1280g2 can also bind non-photoswitching phycoerythrobilin (PEB), resulting in a highly fluorescent protein. Given the small size, high quantum yield, and that unlike green fluorescent proteins, bilin-binding proteins can be used in anaerobic organisms, the orange fluorescent All1280g2-PEB protein is a promising platform for designing new genetically encoded metal ion sensors. Here, we show that All1280g2-PEB undergoes a ∼5-fold reversible zinc-induced fluorescence enhancement with a blue-shifted emission maximum (572 to 517 nm), which is not observed for a related PEB-bound GAF from Synechocystis sp. PCC6803 (Slr1393g3). Zn2+ significantly enhances All1280g2-PEB fluorescence across a biologically relevant pH range from 6.0 to 9.0, with pH-dependent dissociation constants from 1 μM to ∼20-80 nM. Site-directed mutants aiming to sterically decrease and increase access to PEB show a decreased and similar amount of zinc-induced fluorescence enhancement. Mutation of the cysteine residue within the DXCF motif to alanine abolishes the zinc-induced fluorescence enhancement. Collectively, these results support the presence of a unique fluorescence-enhancing Zn2+ binding site in All1280g2-PEB likely involving coordination to the bilin cofactor and requiring a nearby cysteine residue.
摘要：
蓝细菌色素（CBCR）衍生的荧光蛋白是一类可以结合胆色素辅因子并跨紫外线到近红外光谱发出荧光的报告基因。来源于蓝细菌中与植物色素相关的感光蛋白，这些蛋白质中的许多使用单个小GAF结构域来自催化结合bilin并发出荧光。来自Nostocsp的All1280（All1280g2）的第二个GAF结构域。PCC7120是一种含有DXCF基序的蛋白质，当与其天然辅因子结合时，表现出蓝光响应性光化学，藻蓝霉素.All1280g2还可以结合非光开关藻红胆素（PEB），产生高度荧光的蛋白质。鉴于尺寸小，高量子产率，与绿色荧光蛋白不同，bilin结合蛋白可用于厌氧生物，橙色荧光All1280g2-PEB蛋白是设计新的基因编码金属离子传感器的有前途的平台。这里,我们表明，All1280g2-PEB经历了5倍可逆锌诱导的荧光增强与蓝移发射最大值（572至517nm），未观察到来自集胞藻的相关PEB结合的GAF。PCC6803(Slr1393g3)。Zn2+在6.0至9.0的生物学相关pH范围内显着增强All1280g2-PEB荧光，pH依赖性解离常数为1μM至~20-80nM。旨在在空间上减少和增加对PEB的访问的定点突变体显示出减少和相似量的锌诱导的荧光增强。DXCF基序内的半胱氨酸残基突变为丙氨酸消除了锌诱导的荧光增强。总的来说，这些结果支持在All1280g2-PEB中存在独特的荧光增强Zn2+结合位点,可能涉及与胆汁素辅因子的配位并需要附近的半胱氨酸残基.
