Surrey T, Elowitz M B, Wolf P E, Yang F, Nédélec F, Shokat K, Leibler S
Department of Physics, Princeton University, Princeton, NJ 08544, USA.
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4293-8. doi: 10.1073/pnas.95.8.4293.
Chromophore-assisted light inactivation (CALI) offers the only method capable of modulating specific protein activities in localized regions and at particular times. Here, we generalize CALI so that it can be applied to a wider range of tasks. Specifically, we show that CALI can work with a genetically inserted epitope tag; we investigate the effectiveness of alternative dyes, especially fluorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies of pulsed and continuous-wave illumination. We then use fluorescein-labeled hemagglutinin antibody fragments, together with relatively low-power continuous-wave illumination to examine the effectiveness of CALI targeted to kinesin. We show that CALI can destroy kinesin activity in at least two ways: it can either result in the apparent loss of motor activity, or it can cause irreversible attachment of the kinesin enzyme to its microtubule substrate. Finally, we apply this implementation of CALI to an in vitro system of motor proteins and microtubules that is capable of self-organized aster formation. In this system, CALI can effectively perturb local structure formation by blocking or reducing the degree of aster formation in chosen regions of the sample, without influencing structure formation elsewhere.
生色团辅助光灭活（CALI）提供了唯一一种能够在局部区域和特定时间调节特定蛋白质活性的方法。在此，我们对CALI进行了推广，使其能够应用于更广泛的任务。具体而言，我们证明了CALI可以与基因插入的表位标签配合使用；我们研究了替代染料，尤其是荧光素的有效性，并将它们与标准的CALI染料孔雀石绿进行了比较；我们还研究了脉冲光和连续波照明的相对效率。然后，我们使用荧光素标记的血凝素抗体片段，结合相对低功率的连续波照明，来检验靶向驱动蛋白的CALI的有效性。我们表明，CALI至少可以通过两种方式破坏驱动蛋白的活性：它要么导致明显的运动活性丧失，要么导致驱动蛋白酶不可逆地附着在其微管底物上。最后，我们将这种CALI方法应用于一个能够自组织形成星状体的驱动蛋白和微管的体外系统。在这个系统中，CALI可以通过阻止或降低样品选定区域中的星状体形成程度，有效地干扰局部结构的形成，而不会影响其他地方的结构形成。
