Wehler Pierre, Armbruster Daniel, Günter Andreas, Schleicher Erik, Di Ventura Barbara, Öztürk Mehmet Ali
Institute of Biology II, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany.
Centers for Biological Signalling Studies BIOSS and CIBSS, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany.
ACS Omega. 2022 Jun 1;7(23):19555-19560. doi: 10.1021/acsomega.2c01283. eCollection 2022 Jun 14.
iLOV is a flavin mononucleotide-binding fluorescent protein used for cellular imaging similar to the green fluorescent protein. To expand the range of applications of iLOV, spectrally tuned red-shifted variants are desirable to reduce phototoxicity and allow for better tissue penetration. In this report, we experimentally tested two iLOV mutants, iLOV and iLOV, which were previously computationally proposed by (KhrenovaJ. Phys. Chem. B2017, 121 ( (43), ), pp 10018-10025) to have red-shifted excitation and emission spectra. While iLOV is about 20% brighter compared to the WT , it exhibits a blue shift in contrast to quantum mechanics/molecular mechanics (QM/MM) predictions. Additional optical characterization of an iLOV mutant revealed that V392 is essential for cofactor binding and, accordingly, variants with V392K mutation are unable to bind to FMN. iLOV and iLOV are expressed at low levels and have no detectable fluorescence in living cells, preventing their utilization in imaging applications.
iLOV是一种黄素单核苷酸结合荧光蛋白，用于细胞成像，类似于绿色荧光蛋白。为了扩大iLOV的应用范围，需要光谱调谐的红移变体以降低光毒性并实现更好的组织穿透。在本报告中，我们对两个iLOV突变体iLOV和iLOV进行了实验测试，这两个突变体先前由（KhrenovaJ. Phys. Chem. B2017, 121 ( (43), ), pp 10018 - 10025）通过计算预测具有红移的激发和发射光谱。虽然与野生型相比，iLOV的亮度约高20%，但与量子力学/分子力学（QM/MM）预测相反，它表现出蓝移。对一个iLOV突变体的进一步光学表征表明，V392对于辅因子结合至关重要，因此，具有V392K突变的变体无法与FMN结合。iLOV和iLOV在活细胞中的表达水平较低且无可检测的荧光，这使得它们无法用于成像应用。
