Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Poster Abstracts

30-POS Board 18 PIE-FCCS MODEL FOR COMPLEX MEMBRANE HETEROMERS STOICHIOMETRY Mohamed Seghiri 1 ; Adam W Smith 1 ;

1 Texas Tech University, Department of Chemistry and Biochemistry, Lubbock, TX, USA Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) is an advanced fluorescence method that can quantitatively measure biomolecular interactions in living cells. The correlation of fluorescence fluctuations in PIE-FCSS provides information about the density, diffusion, and oligomerization of two-color labeled species. Oligomerization is determined by the amplitude of the correlation functions, the accuracy of which is affected by several factors, including non-overlapping observation volumes, immature and dark states in fluorescent protein labels, photobleaching, and incomplete dimerization. We have previously developed a mathematical model for membrane protein homo-oligomers and compared it with experimental data. However, to account for membrane protein hetero-oligomers, we present a mathematical model describing the different combinations of heteromerization levels and include corrections for non-fluorescent proteins. To validate the model, we used FKBP (FK506-binding protein) and FRB (FKBP rapamycin-binding domain) constructs and induced their heterodimerization with rapamycin. We compared the mathematical model with the experimental data of FRB-FKBP-induced hetero-oligomerization and FKBP-induced homo-oligomerization, showing the relative change in cross-correlation values (f c ). We also assessed the predictive capacity of the mathematical model with respect to the structural organization and binding affinity of these controls.

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