Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.
Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.
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交互式计算和实验方法提高了基于周质结合蛋白的尼古丁生物传感器在生物流体中测量的灵敏度。
DOI：
10.1093/protein/gzae003
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Lester,HenryA
中科院分区：
文献类型：
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作者：
Haloi,Nandan;Huang,Shan;Nichols,AaronL;Fine,EveJ;Friesenhahn,NicholasJ;Marotta,ChristopherB;Dougherty,DennisA;Lindahl,Erik;Howard,RebeccaJ;Mayo,StephenL;Lester,HenryA
我们开发了具有改进灵敏度的尼古丁荧光蛋白传感器。对于pH 7.4的iNicSnFR 12，Δ F/F0与[尼古丁]的比例常数（δ-斜率，2.7 μM−1）比先前报告的iNicSnFR 3a高6.1倍。iNicSnFR 12的活化态具有至少0.6的荧光量子产率。我们测量了尼古丁诱导的吸光度增加和荧光增加的类似剂量-反应关系，表明吸光度增加导致荧光增加，这与先前描述的尼古丁诱导的构象变化，即“蜡烛熄灭”机制无关。分子动力学（MD）模拟确定了尼古丁的结合姿势，以前不确定的实验数据。MD模拟还显示，周质结合蛋白（PBP）结构域的HAP 4在iNicSnFR 12中相对于iNicSnFR 3a似乎倾斜，可能改变了将配体结合位点与荧光团连接的变构网络。在热熔实验中，尼古丁稳定了测试的iNicSnFR变体的PBP。iNicSnFR 12在稀释的小鼠和人血清中以100 nM的浓度解析尼古丁，这是吸烟或吸电子烟期间出现的峰值[尼古丁]，并且可能在会话之间的间隔期间降低水平。NicSnFR 12也被生物流体中未鉴定的内源性配体部分激活。改进的iNicSnFR 12变体可以成为动物和人类生物流体连续尼古丁监测器中的分子传感器。
We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs[nicotine] (δ-slope, 2.7 μM−1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increaseviathe previously described nicotine-induced conformational change, the ‘candle snuffer’ mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.