Gurtovenko Andrey A, Javanainen Matti, Lolicato Fabio, Vattulainen Ilpo
Institute of Macromolecular Compounds, Russian Academy of Sciences , Bolshoi Prospect V.O. 31 , St. Petersburg 199004 , Russia.
Faculty of Physics , St. Petersburg State University , Ulyanovskaya Street 3 , Petrodvorets, St. Petersburg 198504 , Russia.
J Phys Chem Lett. 2019 Mar 7;10(5):1005-1011. doi: 10.1021/acs.jpclett.9b00065. Epub 2019 Feb 19.
Single-particle tracking (SPT) is an experimental technique that allows one to follow the dynamics of individual molecules in biological membranes with unprecedented precision. Given the importance of lipid and membrane protein diffusion in the formation of nanoscale functional complexes, it is critical to understand what exactly is measured in SPT experiments. To clarify this issue, we employed nanoscale computer simulations designed to match SPT experiments that exploit streptavidin-functionalized Au nanoparticles (AuNPs). The results show that lipid labeling interferes critically with the diffusion process; thus, the diffusion measured in SPT is a far more complex process than what has been assumed. It turns out that the influence of AuNP-based labels on the dynamics of probe lipids includes not only the AuNP-induced viscous drag that is the more significant the larger the NP but, more importantly, also the effects related to the interactions of the streptavidin linker with membrane lipids. Due to these effects, the probe lipid moves in a concerted manner as a complex with the linker protein and numerous unlabeled lipids, which can slow down the motion of the probe by almost an order of magnitude. Furthermore, our simulations show that nonlinker streptavidin tetramers on the AuNP surface are able to interact with the membrane lipids, which could potentially lead to multivalent labeling of the NPs by the probe lipids. Our results further demonstrate that in the submicrosecond time domain the motion of the probe lipid is uncorrelated with the motion of the AuNP, showing that there is a 1 μs limit for the temporal resolution of the SPT technique. However, this limit for the temporal resolution depends on the nanoparticle size and increases rapidly with growing AuNPs. Overall, the results provide a molecular-scale framework to accurately interpret SPT data and to design protocols that minimize label-induced artifacts.
单粒子追踪（SPT）是一种实验技术，它能让人们以前所未有的精度追踪生物膜中单个分子的动力学过程。鉴于脂质和膜蛋白扩散在纳米级功能复合物形成中的重要性，了解SPT实验中究竟测量的是什么至关重要。为了阐明这个问题，我们采用了纳米级计算机模拟，旨在匹配利用链霉亲和素功能化金纳米颗粒（AuNP）的SPT实验。结果表明，脂质标记对扩散过程有严重干扰；因此，SPT中测量的扩散是一个比之前假设的要复杂得多的过程。事实证明，基于AuNP的标记对探针脂质动力学的影响不仅包括AuNP引起的粘性阻力（NP越大，这种阻力越显著），更重要的是，还包括与链霉亲和素连接体和膜脂质相互作用相关的影响。由于这些影响，探针脂质与连接体蛋白以及众多未标记的脂质形成复合物，以协同方式移动，这可能会使探针的运动速度减慢近一个数量级。此外，我们的模拟表明，AuNP表面的非连接体链霉亲和素四聚体能够与膜脂质相互作用，这可能会导致探针脂质对NP进行多价标记。我们的结果进一步表明，在亚微秒时间域内，探针脂质的运动与AuNP的运动不相关，这表明SPT技术的时间分辨率存在1微秒的限制。然而，这种时间分辨率的限制取决于纳米颗粒的大小，并且随着AuNP的增大而迅速增加。总体而言，这些结果提供了一个分子尺度的框架，以准确解释SPT数据并设计将标记诱导的伪影降至最低的实验方案。
