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

该奖项支持可用于基于时间分辨磷光各向异性和荧光耗尽各向异性测量细胞膜蛋白质运动的仪器和技术的改进。 采用高脉冲率二极管泵浦Nd：YAG激光器、低后臂时间数字转换器、双通道数据采集和改进的电子设备将使仪器得到改进。 时域测量的检测器门控的改进将探索通过光电倍增管门控的直接光学门控和低后脉冲策略的组合。预计这将允许检查发生在几微秒内的旋转弛豫。将探索荧光损耗各向异性的频域和“连续”版本的实施以检查弱表达的细胞表面蛋白。 将通过在一致的测试系统（即2 H3肥大细胞上的I型Fc受体）上测量蛋白质旋转来评估改进。 测量膜蛋白动力学的能力的提高应该提供对膜结合受体和其他蛋白质的功能的更好理解，这些蛋白质在膜内移动的能力是它们在细胞内信号传导和其他重要生物过程中的作用的关键。

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.