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

Colorado State University, Fort Collins CO

Investigators

Abstract

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.