title: Accurate quantification of diffusion and binding kinetics of non-integral membrane proteins by FRAP.
abstract: Non-integral membrane proteins frequently act as transduction hubs in vital signaling pathways initiated at the plasma membrane (PM). Their biological activity depends on dynamic interactions with the PM, which are governed by their lateral and cytoplasmic diffusion and membrane binding/unbinding kinetics. Accurate quantification of the multiple kinetic parameters characterizing their membrane interaction dynamics has been challenging. Despite a fair number of approximate fitting functions for analyzing fluorescence recovery after photobleaching (FRAP) data, no approach was able to cope with the full diffusion-exchange problem. Here, we present an exact solution and matlab fitting programs for FRAP with a stationary Gaussian laser beam, allowing simultaneous determination of the membrane (un)binding rates and the diffusion coefficients. To reduce the number of fitting parameters, the cytoplasmic diffusion coefficient is determined separately. Notably, our equations include the dependence of the exchange kinetics on the distribution of the measured protein between the PM and the cytoplasm, enabling the derivation of both k(on)  and k(off)  without prior assumptions. After validating the fitting function by computer simulations, we confirm the applicability of our approach to live-cell data by monitoring the dynamics of GFP-N-Ras mutants under conditions with different contributions of lateral diffusion and exchange to the FRAP kinetics.
authors.affiliation: School of Chemistry, Raymond and Beverley Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
authors.forename: Ronen
authors.initials: R
authors.lastname: Berkovich
authors.forename: Haguy
authors.initials: H
authors.lastname: Wolfenson
authors.forename: Sharon
authors.initials: S
authors.lastname: Eisenberg
authors.forename: Marcelo
authors.initials: M
authors.lastname: Ehrlich
authors.forename: Matthias
authors.initials: M
authors.lastname: Weiss
authors.forename: Joseph
authors.initials: J
authors.lastname: Klafter
authors.forename: Yoav I
authors.initials: YI
authors.lastname: Henis
authors.forename: Michael
authors.initials: M
authors.lastname: Urbakh
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D003593:Cytoplasm / Q000378:metabolism; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D011905:Genes, ras; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D015398:Signal Transduction
chemical_list: D008565:Membrane Proteins; D049452:Green Fluorescent Proteins