Quantitative\nAnalysis of Protein–Protein Equilibrium\nConstants in Cellular Environments Using Single-Molecule Localization\nMicroscopy

Current methods for\ndetermining equilibrium constants\noften operate\nin three-dimensional environments, which may not accurately reflect\ninteractions with membrane-bound proteins. With our technique, based\non single-molecule localization microscopy (SMLM), we directly determine\nprotein–protein association (<i>K</i><sub>a</sub>) and dissociation (<i>K</i><sub>d</sub>) constants in\ncellular environments by quantifying associated and isolated molecules\nand their interaction area. We introduce Kernel Surface Density (ks-density,)\na novel method for determining the accessible area for interacting\nmolecules, eliminating the need for user-defined parameters. Simulation\nstudies validate our method’s accuracy across various density\nand affinity conditions. Applying this technique to T cell signaling\nproteins, we determine the 2D association constant of T cell receptors\n(TCRs) in resting cells and the pseudo-3D dissociation constant of\npZAP70 molecules from phosphorylated intracellular tyrosine-based\nactivation motifs on the TCR-CD3 complex. We address challenges of\nmultiple detection and molecular labeling efficiency. This method\nenhances our understanding of protein interactions in cellular environments,\nadvancing our knowledge of complex biological processes.