Transmembrane proteins play important roles in intercellular signaling to regulate interactions among the adjacent cells and influence cell fate. The study of interactions between membrane proteins and nanomaterials is paramount for the design of nanomaterial-based therapies. In the present work, the fluorescence properties of the transmembrane receptor Notch2 have been investigated. In particular, the steady-state and time-resolved fluorescence methods have been used to characterize the emission of tryptophan residues of Notch2 and then this emission is used to monitor the effect of silver colloids on protein behavior. To this aim, silver colloids are prepared with two different methods to make sure that they bear hydrophilic (citrate ions, C-AgNPs) or hydrophobic (dodecanethiol molecules, D-AgNPs) capping agents. The preparation procedures are tightly controlled to obtain metal cores with similar size distributions (7.4 ± 2.5 and 5.0 ± 0.8 nm, respectively), thus, making the comparison of the results easier. The occurrence of strong interactions between Notch2 and D-AgNPs is suggested by the efficient and statistically relevant quenching of the stationary protein emission already at low nanoparticle (NP) concentrations (ca. 12% quenching with [D-AgNPs] = 0.6 nM). The quenching becomes even more pronounced (ca. 60%) when [D-AgNPs] is raised to 8.72 nM. On the other hand, the addition of increasing concentrations of C-AgNPs to Notch2 does not affect the protein fluorescence (intensity variations below 5%) indicating that negligible interactions are taking place. The fluorescence data, recorded in the presence of increasing concentrations of silver nanoparticles, are then analyzed through the Stern–Volmer equation and the sphere of action model to discuss the nature of interactions. The effect of D-AgNPs on the fluorescence decay times of Notch2 is also investigated and a decrease in the average decay time is observed (from 4.64 to 3.42 ns). The observed variations of the stationary and time-resolved fluorescence behavior of the protein are discussed in terms of static and collisional interactions. These results document that the capping shell is able to drive the protein–particle interactions, which likely have a hydrophobic nature.

中文翻译：

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跨膜蛋白的荧光分析及其与功能不同的银纳米粒子的相互作用

跨膜蛋白在细胞间信号传导中起着重要作用，以调节相邻细胞之间的相互作用并影响细胞命运。膜蛋白与纳米材料之间相互作用的研究对于基于纳米材料的疗法的设计至关重要。在目前的工作中，已经研究了跨膜受体Notch2的荧光性质。特别是，稳态和时间分辨荧光方法已用于表征Notch2色氨酸残基的发射，然后将该发射用于监测银胶体对蛋白质行为的影响。为此，可通过两种不同方法制备银胶体，以确保它们带有亲水性（柠檬酸根离子，C-AgNPs）或疏水性（十二烷硫醇分子，D-AgNPs）封端剂。严格控制制备过程，以获得具有相似尺寸分布（分别为7.4±2.5和5.0±0.8 nm）的金属芯，从而使结果的比较更加容易。Notch2和D-AgNP之间强烈相互作用的发生是由于在低纳米颗粒（NP）浓度下已进行的固定蛋白质发射的有效且统计上相关的淬灭（[D-AgNPs = 0.6 nM时约为12％淬灭）。当[D-AgNPs]升高至8.72 nM时，猝灭甚至更加明显（约60％）。另一方面，在Notch2中添加浓度不断增加的C-AgNPs不会影响蛋白质荧光（强度变化低于5％），表明发生的相互作用可忽略不计。荧光数据 在银纳米颗粒浓度不断增加的情况下记录的碳，然后通过Stern-Volmer方程和作用域模型进行分析，以讨论相互作用的性质。还研究了D-AgNPs对Notch2荧光衰减时间的影响，并观察到平均衰减时间的减少（从4.64 ns减少到3.42 ns）。从静态和碰撞相互作用的角度讨论了蛋白质的固定和时间分辨荧光行为的观察到的变化。这些结果证明，封盖壳能够驱动蛋白质-颗粒相互作用，这可能具有疏水性。还研究了D-AgNPs对Notch2荧光衰减时间的影响，并观察到平均衰减时间的减少（从4.64 ns减少到3.42 ns）。从静态和碰撞相互作用的角度讨论了蛋白质的固定和时间分辨荧光行为的观察到的变化。这些结果证明，封盖壳能够驱动蛋白质-颗粒相互作用，这可能具有疏水性。还研究了D-AgNPs对Notch2荧光衰减时间的影响，并观察到平均衰减时间的减少（从4.64 ns减少到3.42 ns）。从静态和碰撞相互作用的角度讨论了蛋白质的固定和时间分辨荧光行为的观察到的变化。这些结果证明，封盖壳能够驱动蛋白质-颗粒相互作用，这可能具有疏水性。