组成图示
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传感器类型
其他（NADPH依赖蓝色荧光蛋白/FRET供体研究）
检测对象
NADPH（NADPH，还原型烟酰胺腺嘌呤二核苷酸磷酸）；样品基质：纯化蛋白溶液/大肠杆菌表达体系（未报告血清、尿液等实际样品基质；氧仅为潜在传感对象）
检测原理
BFPvvD8作为短链脱氢酶/还原酶家族蛋白，其活性位点结合NADPH。NADPH本身荧光弱且寿命短（0.43 ns），结合后被蛋白微环境屏蔽并刚性化，在352 nm激发下发射440 nm蓝光。G176S突变改变PG box附近主链构象，使A177主链N与NADPH烟酰胺O7接近，形成电荷/氢键相互作用，限制烟酰胺环与酰胺基团间C-C单键扭转，降低非辐射衰减，延长寿命（WT 4.29 ns，G176S 7.26 ns，D8 7.03 ns），从而提高发射强度。融合蛋白中，BFPvvD8作为FRET供体，mTFP1或EGFP作为受体，供体发射与受体吸收重叠，能量非辐射转移后产生493/510 nm绿光。NADPH结合状态或浓度改变供体发射和FRET效率；未使用外源放大策略，主要依赖蛋白刚性化与FRET。
检测灵敏度
原文未报告LOD、线性范围、灵敏度斜率或相关系数。
效应效果
性能方面，BFPvvD8相对D7发射强度提高27%（D7 100.00%±1.06%，D8 126.86%±1.39%），相对野生型约4倍（WT 25.28%±1.81%，D8 100.00%±2.87%）。G176S/G176A使野生型发射提高约2倍，发射峰由约450 nm蓝移至440 nm。荧光寿命由NADPH单独0.43 ns延长至WT-NADPH 4.29 ns、G176S-NADPH 7.26 ns、D8-NADPH 7.03 ns。ITC显示WT与D8对NADPH结合常数相近，增强并非来自亲和力提高。BFPvvD8缺氧下仍可发光，EGFP不能。未报告选择性、抗干扰、稳定性、RSD、实际样品回收率或方法对比，但作者主张其可作FRET型NADPH/氧传感器潜在供体。
传感器的构成
- 荧光换能/供体：BFPvvD8，NADPH依赖蓝色荧光蛋白，结合NADPH后发射440 nm蓝光
- 识别/结合元件：BFPvvD8活性位点，结合NADPH并受G176S突变影响
- 辅因子/内源发色团：NADPH，作为发色团，结合后增强荧光并蓝移
- 荧光受体元件：mTFP1或EGFP，GFP样蛋白，接受FRET能量后发射493/510 nm绿光
- 连接子：HVVDDDDK，8残基肽段，连接BFPvvD8 N端与GFP C端
- 表达载体：pET46 Ek/LIC，用于构建BFPvvD8-GFP融合蛋白
- 表达宿主：BL21(DE3)大肠杆菌，IPTG诱导表达融合蛋白
- 读出仪器：Fluorolog-3、LS-55或nF900-M300，记录发射光谱与寿命
中文摘要
本文报道了来自副溶血弧菌CKM-1的NADPH依赖蓝色荧光蛋白BFPvvD8的结构与荧光增强机制。BFPvv在紫外光激发下发射蓝色荧光，此前通过定向进化获得的BFPvvD7突变体荧光强度提高约4倍。作者进一步在D7基础上引入N58Y、A62T和K199R三个突变，获得荧光更强的BFPvvD8。为阐明增强机制，解析了BFPvvD8与NADPH复合物的晶体结构，分辨率为2.05 Å。结合荧光寿命检测，作者提出荧光增强与NADPH结合位点附近转角处Gly176突变为非甘氨酸残基引起的构象变化有关：该突变使主链扭转，使邻近残基与NADPH烟酰胺基团形成相互作用，限制发色团运动并降低非辐射衰减。此外，作者构建了BFPvvD8与mTFP1或EGFP的融合蛋白，观察到BFPvvD8向两种GFP样受体蛋白的Förster共振能量转移。结果表明，BFPvvD8不仅为理性蛋白工程提供结构依据，也可作为FRET型生物传感器技术的潜在候选供体。
英文摘要
A NADPH-dependent blue fluorescent protein from Vibrio vulnificus CKM-1 (BFPvv) emits blue fluorescence under UV-exposure. Previously, the BFPvvD7 mutant generated by directed evolution displayed a fourfold enhancement in fluorescent intensity. Herein, a further increase in fluorescence in the new BFPvvD8 mutant, with three additional mutations from BFPvvD7, was made. To understand the underlying mechanism of the increased fluorescent intensity of BFPvv, we solved the BFPvvD8-NADPH complex structure. Accompanied with lifetime detection, we proposed that the enhanced intensity is related to the conformational change caused by a glycine residue (Gly176) mutated to other non-glycine residues at a turn close to the NADPH binding site. We also observed the Förster resonance energy transfer (FRET) from our BFPvvD8 to each of the GFP-like fluorescent proteins, mTFP1 and EGFP, joined by an eight-residue linker between the N-terminal of BFPvvD8 and the C-terminal of GFPs. Taken together, with the newly solved BFPvvD8 structure, our results not only provide new considerations within the rational-based protein engineering of this NADPH-dependent BFP, but also suggest that BFPvvD8 could be a potential candidate in FRET-based biosensor techniques.