A novel single-molecule study to determine protein-protein association constants
A novel single-molecule study to determine protein-protein association constants
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DOI：
10.1021/ja005750n
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发表时间：
2001-06-20
影响因子：
15
通讯作者：
Erie, DA
中科院分区：
文献类型：
--
作者：
Ratcliff, GC;Erie, DA
原子力显微镜（AFM）传统上被用作一种成像技术来获得生物系统的定性信息。我们已经成功地利用AFM的成像能力来确定蛋白质-蛋白质结合常数。我们已经开发了一种方法来测量蛋白质的分子量基于其从原子力显微镜图像确定的体积。我们的体积测定方法允许快速，准确地分析大量蛋白质群体。在测量体积的基础上，确定了DNA解旋酶UvrD的单体占二聚体的比例，并计算了解旋酶的解离常数K-d。我们确定UvrD的K-d为1.4 muM，这与从分析性超离心（AUC）研究中获得的已发表的K-d数据很好地一致。我们的方法提供了一种快速测定蛋白质-蛋白质结合常数的方法。
Atomic force microscopy (AFM) is traditionally used as an imaging technique to gain qualitative information for a biological system. We have successfully used the imaging capabilities of the AFM to determine protein-protein association constants. We have developed a method to measure the molecular weight of a protein based on its volume determined from AFM images. Our volume determination method allows for rapid, accurate analysis of large protein populations. On the basis of the measured volume, the fraction of monomers as dimers was determined for the DNA helicase UvrD, and the dissociation constant (K-d) for the helicase was calculated. We determined a K-d for UvrD of 1.4 muM, which is in good agreement with published K-d data obtained from analytical ultracentrifugation (AUC) studies. Our method provides a rapid method for determining protein-protein association constants.