Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Biophys J. 2010 Nov 3;99(9):2737-47. doi: 10.1016/j.bpj.2010.09.013.
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.
大多数发生在细胞中的重要相互作用类型可以被描述为结合-扩散类型的过程，并可以通过动力学速率常数来量化，如扩散系数 (D) 和结合速率常数 (k(on) 和 k(off))。共焦荧光漂白恢复 (FRAP) 是定量分析细胞内结合-扩散动力学的一种潜在重要工具，但如何从这些分析中可靠地提取准确的动力学常数仍然是一个悬而未决的问题。为此，在本研究中，我们开发了一种我们认为是基于封闭形式的 FRAP 公式的共焦 FRAP 测量的新分析模型，用于测量细胞内的结合-扩散过程。该方法结合了一个结合扩散模型，允许未结合和结合的物质扩散，并且还补偿了在光漂白过程中发生的结合扩散，这是共焦 FRAP 分析中的一个关键考虑因素。此外，为了解决参数多重性问题，我们提出了一种方案，当知道结合和未结合物质的 D 值时，在有效扩散子区域中减少拟合参数的数量。我们通过测量 CAAX 介导的 Ras 与内质网膜结合的动力学速率常数来验证这种方法，得到了 k(on) ∼ 255/s 和 k(off) ∼ 31/s 的结合常数。
