These authors contributed equally to this work.
Accurate Quantification of Diffusion and Binding Kinetics of Non-integral Membrane Proteins by FRAP
Article first published online: 30 AUG 2011
© 2011 John Wiley & Sons A/S
Volume 12, Issue 11, pages 1648–1657, November 2011
How to Cite
Berkovich, R., Wolfenson, H., Eisenberg, S., Ehrlich, M., Weiss, M., Klafter, J., Henis, Y. I. and Urbakh, M. (2011), Accurate Quantification of Diffusion and Binding Kinetics of Non-integral Membrane Proteins by FRAP. Traffic, 12: 1648–1657. doi: 10.1111/j.1600-0854.2011.01264.x
- Issue published online: 6 OCT 2011
- Article first published online: 30 AUG 2011
- Accepted manuscript online: 2 AUG 2011 07:37AM EST
- Received 19 May 2011, revised and accepted for publication 1 August 2011, uncorrected manuscript published online 2 August 2011, published online 30 August 2011
- binding kinetics;
- diffusion analysis;
- membrane interactions;
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 kon and koff 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.