Centro de Investigaciones en Bionanociencias - "Elizabeth Jares-Erijman" (CIBION), CONICET, Godoy Cruz 2390, 1425 Ciudad de Buenos Aires, Argentina.
London Centre for Nanotechnology, University College London, 19 Gordon Street, WC1H 0AH London, United Kingdom.
Nano Lett. 2024 Oct 30;24(43):13834-13842. doi: 10.1021/acs.nanolett.4c04394. Epub 2024 Oct 21.
Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein-protein association () and dissociation () constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method's accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.
目前用于确定平衡常数的方法通常在三维环境中进行，这可能无法准确反映与膜结合蛋白的相互作用。我们的技术基于单分子定位显微镜（SMLM），通过量化相关和分离的分子及其相互作用面积，直接在细胞环境中确定蛋白质-蛋白质的缔合（）和解离（）常数。我们引入了核表面密度（ks-density，）这是一种确定相互作用分子可及面积的新方法，消除了对用户定义参数的需求。模拟研究验证了我们的方法在各种密度和亲和力条件下的准确性。我们将该技术应用于 T 细胞信号蛋白，确定了静止细胞中 T 细胞受体（TCR）的 2D 缔合常数，以及 TCR-CD3 复合物上磷酸化的胞内酪氨酸基激活基序上 pZAP70 分子的拟 3D 解离常数。我们解决了多个检测和分子标记效率的挑战。这种方法增强了我们对细胞环境中蛋白质相互作用的理解，促进了我们对复杂生物过程的认识。
