Uncoupling diffusion and binding in FRAP experiments.
Uncoupling diffusion and binding in FRAP experiments.
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FRAP 实验中解偶联扩散和结合。
DOI：
10.1038/nmeth0309-183a
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发表时间：
2009
期刊：
影响因子：
48
通讯作者：
Lambert,NevinA
中科院分区：
文献类型：
--
作者：
Lambert,NevinA
致编辑：在他们最近的文章中，Schwarzenbacher等人1使用了一种优雅的微图案技术来证明固定的CD 4受体对相互作用蛋白Lck的移动性的影响。蛋白质固定化方法的一个强大的功能是能够提取结合和非结合参数使用荧光恢复后光漂白（FRAP）2，3。然而，在计算CD 4-Lck解结合率时，作者做了一个简化的假设，这引入了严重的错误风险：如果结合事件足够频繁和持久，荧光分子与固定位点的结合将减缓光漂白后的恢复。结合对FRAP的影响取决于结合和扩散的相对速率。在扩散比结合快得多的极端情况下，荧光恢复仅反映未结合。这些情况被称为扩散解耦，以区别于荧光恢复是结合和扩散（扩散耦合）的复杂函数的情况3，4。Schwarzenbacher等人1在得出Lck对比度恢复是扩散解耦的结论后，推导出CD 4-Lck相互作用的寿命。他们在比较了Lck分子扩散穿过漂白区域所需的时间（特征扩散时间）和恢复荧光对比度所需的时间后得出了这一结论。全长Lck（扩散系数约为0.2-1.0 µm2 s-1）穿过20× 5 µm矩形的扩散时间约为6-30 s。作者认为，与光漂白后恢复所需的160 s相比，这是足够快的，扩散可以忽略不计，因此恢复速率对应于未结合速率。虽然这具有直观的意义，但不能简单地通过比较扩散和恢复速率来区分扩散耦合和扩散非耦合机制4。一个必要的初步步骤是根据经验确定扩散是否对复苏产生影响。这通常通过测量作为漂白区域大小的函数的恢复来完成。
To the Editor: In their recent article, Schwarzenbacher et al. 1 use an elegant micropatterning technique to demonstrate the effect of immobile CD4 receptors on the mobility of the interacting protein Lck. One powerful feature of protein immobilization methods is the ability to extract binding and unbinding parameters using fluorescence recovery after photobleaching (FRAP) 2, 3. However, in their calculation of CD4-Lck unbinding rates, the authors made a simplifying assumption that introduces a serious risk of error.Binding of fluorescent molecules to immobile sites will slow recovery after photobleaching provided the binding events are sufficiently frequent and long-lived. The effect of binding on FRAP depends on the relative rates of binding and diffusion. At the extreme where diffusion is much more rapid than binding, fluorescence recovery reflects only unbinding. These circumstances are referred to as diffusion-uncoupled, to distinguish them from instances in which fluorescence recovery is a complex function of both binding and diffusion (diffusion-coupled) 3, 4. Schwarzenbacher et al. 1 derive the lifetime of the CD4-Lck interaction after concluding that Lck contrast recovery is diffusion-uncoupled. They made this conclusion after comparing the time required for an Lck molecule to diffuse across a bleached region (the characteristic diffusion time) and the time required for recovery of fluorescence contrast. The diffusion time for full-length Lck (with a diffusion coefficient of~ 0.2–1.0 µm2 s-1) across 20× 5 µm rectangles will be~ 6–30 s. The authors argue that this is sufficiently rapid compared to the 160 s required for recovery after photobleaching that diffusion is negligible and thus that the recovery rate corresponds to the unbinding rate. Although this makes intuitive sense, the diffusion-coupled and diffusion-uncoupled regimes cannot be distinguished simply by comparing diffusion and recovery rates4. A necessary preliminary step is to determine empirically whether diffusion has an impact on recovery. This is most commonly done by measuring recovery as a function of bleached region size.