Abstract:
: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.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Kang M,Day CA,DiBenedetto E,Kenworthy AKdoi
10.1016/j.bpj.2010.09.013subject
Has Abstractpub_date
2010-11-03 00:00:00pages
2737-47issue
9eissn
0006-3495issn
1542-0086pii
S0006-3495(10)01114-8journal_volume
99pub_type
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