| Autor: |
Marcano-García LF; Centro de Investigaciones en Bionanociencias - 'Elizabeth Jares-Erijman' (CIBION), CONICET, Godoy Cruz 2390, 1425 Ciudad de Buenos Aires, Argentina., Zaza C; London Centre for Nanotechnology, University College London, 19 Gordon Street, WC1H 0AH London, United Kingdom., Dalby OPL; London Centre for Nanotechnology, University College London, 19 Gordon Street, WC1H 0AH London, United Kingdom.; Department of Chemistry, University College London, 20 Gordon Street, WC1H 0AJ London, United Kingdom., Joseph MD; London Centre for Nanotechnology, University College London, 19 Gordon Street, WC1H 0AH London, United Kingdom.; Department of Chemistry, University College London, 20 Gordon Street, WC1H 0AJ London, United Kingdom., Cappellari MV; Centro de Investigaciones en Bionanociencias - 'Elizabeth Jares-Erijman' (CIBION), CONICET, Godoy Cruz 2390, 1425 Ciudad de Buenos Aires, Argentina., Simoncelli S; London Centre for Nanotechnology, University College London, 19 Gordon Street, WC1H 0AH London, United Kingdom.; Department of Chemistry, University College London, 20 Gordon Street, WC1H 0AJ London, United Kingdom., Aramendía PF; Centro de Investigaciones en Bionanociencias - 'Elizabeth Jares-Erijman' (CIBION), CONICET, Godoy Cruz 2390, 1425 Ciudad de Buenos Aires, Argentina. |
| Abstrakt: |
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 ( K a ) and dissociation ( K d ) 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. |
