Analysis of membrane-localized binding kinetics with FRAP.
Interactions between plasma membrane-associated proteins on interacting cells are critical for many important biological processes. Few experimental techniques, however, can accurately determine the association and the dissociation rates between such interacting pairs when the two molecules diffuse on apposing membranes or lipid bilayers. In this study, we give a theoretical description of how and when fluorescence recovery after photobleaching (FRAP) experiments can be used to quantify these reaction rates. We analyze the effect of binding on FRAP recovery curves with a reaction-diffusion model and systematically identify different regimes in the parameter space of the association and the dissociation constants for which the full model simplifies into equivalent one-parameter models. Based on this analysis, we propose an experimental protocol that may be used to identify the kinetic parameters of binding in the appropriate parameter regime. We present simulated experiments illustrating our protocol and lay down guidelines for parameter estimation.
Dushek
Omer
O
Department of Mathematics, University of British Columbia, Vancouver, BC V6T 1Z2, Canada.
Das
Raibatak
R
Coombs
Daniel
D
D002462:Cell Membrane / Q000378:metabolism*; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D011485:Protein Binding; D017186:Titrimetry
D008024:Ligands