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The estimation of binding constants and diffusion coefficients of molecules that associate with insoluble molecular scaffolds inside living cells and nuclei has been facilitated by the use of Fluorescence Recovery after Photobleaching (FRAP) in conjunction with mathematical modeling. A critical feature unique to FRAP experiments that has been overlooked by past mathematical treatments is the existence of an 'equilibrium constraint': local dynamic equilibrium is not disturbed because photobleaching does not functionally destroy molecules, and hence binding-unbinding proceeds at equilibrium rates. Here we describe an improved mathematical formulation under the equilibrium constraint which provides a more accurate estimate of molecular reaction kinetics within FRAP studies carried out in living cells. Due to incorporation of the equilibrium constraint, the original nonlinear kinetic terms become linear allowing for analytical solution of the transport equations and greatly simplifying the estimation process. Based on mathematical modeling and scaling analysis, two experimental measures are identified that can be used to delineate the rate-limiting step. A comprehensive analysis of the interplay between binding-unbinding and diffusion, and its effect on the recovery curve, are presented. This work may help to bring clarity to the study of molecular dynamics within the structural complexity of living cells.

An improved mathematical approach for determination of molecular kinetics in living cells with FRAP.