Improved Instrumentation and Procedures for Time-Resolved Phosphorescence Anisotropy and Fluorescence Depletion Anisotropy Measurements of Membrane Protein Rotation

该奖项支持改进可用于基于时间分辨磷光各向异性和荧光耗尽各向异性来测量细胞膜蛋白质运动的仪器和技术。仪器的改进将源于引入高脉冲速率的二极管泵浦的Nd：YAG激光器、低再武装时间的数字化仪、双通道数据采集和改进的电子学。通过组合直接光学选通和用于光电倍增管选通的低脉冲后脉冲策略，将探索用于时域测量的探测器选通的改进。预计这将允许检查发生在几微秒内的旋转松弛。对荧光耗竭各向异性的频域和“连续”版本的实现将被探索用于检测弱表达的细胞表面蛋白。改进将通过在一致的测试系统上测量蛋白质旋转来评估，即2H3肥大细胞上的I型Fc受体。膜蛋白动态测量能力的提高应该有助于更好地了解膜结合受体和其他蛋白质的功能，这些蛋白质在膜内移动的能力是它们在细胞内信号传递和其他重要生物过程中作用的关键。

This award supports the improvement of instrumentation and techniques that can be used for measurement of movement of cell membrane proteins based on time-resolved phosphorescence anisotropy and fluorescence depletion anisotropy. Improved instrumentation will result from introduction of a high pulse rate diode-pumped Nd:YAG laser, low rearm-time digitizers, two-channel data acquisition and improved electronics. Improvements in detector gating for time-domain measurements will be explored by a combination of direct optical gating and low after-pulsing strategies for photomultiplier gating. This is expected to permit examination of rotational relaxations occurring within a few microseconds. The implementation of frequency-domain and "continuous" versions of fluorescence depletion anisotropy will be explored for examination of weakly- expressed cell surface proteins. Improvements will be evaluated by measuring protein rotation on a consistent test system, namely the Type I Fc receptor on 2H3 mast cells. Improvements in the ability to measure the dynamics of membrane proteins should provide greater understanding of the function of membrane-bound receptors and other proteins whose ability to move within the membrane is key to their role in intracellular signaling and other important biological processes.