Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
J Struct Biol. 2011 Jun;174(3):485-93. doi: 10.1016/j.jsb.2011.02.010. Epub 2011 Mar 21.
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.
一种来自创伤弧菌（Vibrio vulnificus CKM-1）的依赖 NADPH 的蓝色荧光蛋白（BFPvv）在紫外光照射下发出蓝色荧光。此前，通过定向进化产生的 BFPvvD7 突变体显示出荧光强度增强了四倍。在此，通过在 BFPvvD7 上添加三个额外突变，进一步产生了新的 BFPvvD8 突变体，其荧光强度进一步增强。为了了解 BFPvv 荧光强度增强的潜在机制，我们解析了 BFPvvD8-NADPH 复合物的结构。通过寿命检测，我们提出，增强的强度与由靠近 NADPH 结合位点的转角处的甘氨酸残基（Gly176）突变为其他非甘氨酸残基引起的构象变化有关。我们还观察到 BFPvvD8 与通过 BFPvvD8 的 N 端和 GFP 的 C 端之间的 8 个残基连接子连接的 GFP 样荧光蛋白 mTFP1 和 EGFP 之间的Förster 共振能量转移（FRET）。总之，结合新解析的 BFPvvD8 结构，我们的结果不仅为这种依赖 NADPH 的 BFP 的基于理性的蛋白质工程提供了新的考虑因素，而且还表明 BFPvvD8 可能是基于 FRET 的生物传感器技术的潜在候选者。
