Abstract

BackgroundQuantification of in-vivo biomolecule mass transport and reaction rate parameters from experimental data obtained by Fluorescence Recovery after Photobleaching (FRAP) is becoming more important.Methods and resultsThe Osborne-Moré extended version of the Levenberg-Marquardt optimization algorithm was coupled with the experimental data obtained by the Fluorescence Recovery after Photobleaching (FRAP) protocol, and the numerical solution of a set of two partial differential equations governing macromolecule mass transport and reaction in living cells, to inversely estimate optimized values of the molecular diffusion coefficient and binding rate parameters of GFP-tagged glucocorticoid receptor. The results indicate that the FRAP protocol provides enough information to estimate one parameter uniquely using a nonlinear optimization technique. Coupling FRAP experimental data with the inverse modeling strategy, one can also uniquely estimate the individual values of the binding rate coefficients if the molecular diffusion coefficient is known. One can also simultaneously estimate the dissociation rate parameter and molecular diffusion coefficient given the pseudo-association rate parameter is known. However, the protocol provides insufficient information for unique simultaneous estimation of three parameters (diffusion coefficient and binding rate parameters) owing to the high intercorrelation between the molecular diffusion coefficient and pseudo-association rate parameter. Attempts to estimate macromolecule mass transport and binding rate parameters simultaneously from FRAP data result in misleading conclusions regarding concentrations of free macromolecule and bound complex inside the cell, average binding time per vacant site, average time for diffusion of macromolecules from one site to the next, and slow or rapid mobility of biomolecules in cells.ConclusionTo obtain unique values for molecular diffusion coefficient and binding rate parameters from FRAP data, we propose conducting two FRAP experiments on the same class of macromolecule and cell. One experiment should be used to measure the molecular diffusion coefficient independently of binding in an effective diffusion regime and the other should be conducted in a reaction dominant or reaction-diffusion regime to quantify binding rate parameters. The method described in this paper is likely to be widely used to estimate in-vivo biomolecule mass transport and binding rate parameters.

Highlights

  • Quantification of in-vivo biomolecule mass transport and reaction rate parameters from experimental data obtained by Fluorescence Recovery after Photobleaching (FRAP) is becoming more important

  • One experiment should be used to measure the molecular diffusion coefficient independently of binding in an effective diffusion regime and the other should be conducted in a reaction dominant or reaction-diffusion regime to quantify binding rate parameters

  • The method described in this paper is likely to be widely used to estimate in-vivo biomolecule mass transport and binding rate parameters

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Summary

Introduction

Quantification of in-vivo biomolecule mass transport and reaction rate parameters from experimental data obtained by Fluorescence Recovery after Photobleaching (FRAP) is becoming more important. One of the most widely used techniques for studying in vitro and in vivo diffusion and binding reactions, nuclear protein mobility, solute and biomolecule transport through cell membranes, lateral diffusion of lipids in cell membranes, and biomolecule diffusion within the cytoplasm and nucleus, is Fluorescence Recovery after Photobleaching (FRAP). The technique was developed in the 1970s and was initially used to study lateral diffusion of lipids through the cell membrane [1,2,3,4,5,6,7,8,9]. A detailed description of the protocol is presented in [13,15]

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