Abstract：
Binding of fluorescent ligand (FL) to the cyan fluorescent protein (CFP)-coupled ligand-binding domain of the inositol 1,4,5-trisphosphate (IP3) receptor (CFP-LBP) produces fluorescence (Förster) resonance energy transfer (FRET). A competitive fluorescent ligand assay (CFLA), using the FRET signal from competition between FLs and IP3, can measure IP3 concentration. The FRET signal should be enhanced by attaching a FRET donor to an appropriate position. Herein, we inserted five different circularly permuted CFPs in the loop between the second and third α-helices to generate membrane-targeted fluorescent ligand-binding proteins (LBPs). Two such proteins, LBP-cpC157 and LBP-cpC173, localized at the plasma membrane, displayed FRET upon binding the high-affinity ligand fluorescent adenophostin A (F-ADA), and exhibited a decreased fluorescence emission ratio (480 nm / 535 nm) by 1.6- to 1.8-fold that of CFP-LBP. In addition, binding of a fluorescent low-affinity ligand (F-LL) also reduced the fluorescence ratio in a concentration-dependent manner, with EC50 values for LBP-cpC157 and LBP-cpC173 of 34.7 nM and 27.6 nM, respectively. These values are comparable to that with CFP-LBP (29.2 nM), indicating that insertion of cpC157 and cpC173 did not disrupt LBP structure and function. The effect of 100 nM F-LL on the decrease in fluorescence ratio was reversed upon addition of IP3, indicating binding competition between F-LL and IP3. We also constructed cytoplasmic fluorescent proteins cyLBP-cpC157 and cyLBP-cpC173, and bound them to DYK beads for imaging analyses. Application of F-ADA decreased the fluorescence ratio of the beads from the periphery to the center over 3 - 5 min. Application of F-LL also decreased the fluorescence ratio of cyLBP-cpC157 and cyLBP-cpC173 by 20-25%, and subsequent addition of IP3 recovered the fluorescence ratio in a concentration-dependent manner. The EC50 value and Hill coefficient obtained by curve fitting against the IP3-dependent recovery of fluorescence ratio can be used to estimate the IP3 concentration.
摘要：
荧光配体（FL）与肌醇1,4,5-三磷酸（IP3）受体（CFP-LBP）的青色荧光蛋白（CFP）偶联的配体结合域的结合产生荧光（Förster）共振能量转移（FRET）。竞争性荧光配体测定（CFLA），利用来自FL和IP3之间竞争的FRET信号,可以测量IP3浓度。FRET信号应该通过将FRET供体连接到适当的位置来增强。在这里，我们在第二和第三α螺旋之间的环中插入了五个不同的环状置换CFP，以生成膜靶向荧光配体结合蛋白（LBP）。两种这样的蛋白质，LBP-cpC157和LBP-cpC173，位于质膜，在结合高亲和力配体荧光腺样体A（F-ADA）时显示FRET，荧光发射比(480nm/535nm)比CFP-LBP降低1.6-1.8倍。此外,荧光低亲和力配体（F-LL）的结合也以浓度依赖性方式降低了荧光比，LBP-cpC157和LBP-cpC173的EC50值为34.7nM和27.6nM，分别。这些值与CFP-LBP（29.2nM）相当，表明cpC157和cpC173的插入不会破坏LBP的结构和功能。在添加IP3时,100nMF-LL对荧光比率降低的影响被逆转,表明F-LL和IP3之间的结合竞争。我们还构建了细胞质荧光蛋白cyLBP-cpC157和cyLBP-cpC173，并将它们结合到DYK珠子上进行成像分析。F-ADA的应用在3-5分钟内降低了珠子从外围到中心的荧光比率。F-LL的应用还将cyLBP-cpC157和cyLBP-cpC173的荧光比降低了20-25％，并且随后加入IP3以浓度依赖性方式恢复了荧光比。通过对IP3依赖性荧光回收率的曲线拟合获得的EC50值和Hill系数可用于估计IP3浓度。
