Marcano-García, Luis F; Zaza, Cecilia; Dalby, Olivia PL; Joseph, Megan D; Cappellari, M Victoria; Simoncelli, Sabrina; Aramendía, Pedro F; (2024) Quantitative Analysis of Protein-Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy. Nano Letters , 24 (43) pp. 13834-13842. 10.1021/acs.nanolett.4c04394.

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Abstract

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 (Ka) and dissociation (Kd) 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.

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