A quantitative approach to analyze binding diffusion kinetics by confocal FRAP.
Most of the important types of interactions that occur in cells can be characterized as binding-diffusion type processes, and can be quantified by kinetic rate constants such as diffusion coefficients (D) and binding rate constants (k(on) and k(off)). Confocal FRAP is a potentially important tool for the quantitative analysis of intracellular binding-diffusion kinetics, but how to dependably extract accurate kinetic constants from such analyses is still an open question. To this end, in this study, we developed what we believe is a new analytical model for confocal FRAP-based measurements of intracellular binding-diffusion processes, based on a closed-form equation of the FRAP formula for a spot photobleach geometry. This approach incorporates a binding diffusion model that allows for diffusion of both the unbound and bound species, and also compensates for binding diffusion that occurs during photobleaching, a critical consideration in confocal FRAP analysis. In addition, to address the problem of parametric multiplicity, we propose a scheme to reduce the number of fitting parameters in the effective diffusion subregime when D's for the bound and unbound species are known. We validate this method by measuring kinetic rate constants for the CAAX-mediated binding of Ras to membranes of the endoplasmic reticulum, obtaining binding constants of k(on) ∼ 255/s and k(off) ∼ 31/s.
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Minchul
M
Kang
Charles A
CA
Day
Emmanuele
E
DiBenedetto
Anne K
AK
Kenworthy
D000818:Animals; D055592:Biophysical Phenomena; D019556:COS Cells; D002522:Chlorocebus aethiops; D004058:Diffusion; D004721:Endoplasmic Reticulum / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods* / Q000706:statistics & numerical data; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D038761:Photobleaching; D011485:Protein Binding; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016283:Proto-Oncogene Proteins p21(ras)