Abstract
MFD (multiparameter fluorescence detection) is based on simultaneous recording of fluorescence lifetime, polarization, and color. hpFRET (high precision Forster resonance energy transfer) used in single-molecule experiment can show an existence of diverse conformations adopted by macromolecules.fFCS (filtered Fluorescence Correlation Spectroscopy) is extended analysis method of time, polarization and color resolved FCS. The method uses MFD data to obtain information on the kinetics of transitions between fluorescence species on the multiple timescales (ns to ms).The above techniques were applied to hGBP1 (the human guanylate binding protein 1) which is a GTP binding interferon-g induced protein important immuno defense. hGBP1 consists of three domains: a GTP-binding domain (gray), a middle domain (blue) and a helical domain (green). In solution it undergoes conformational transitions between a minor and a major state. The timescale of motion and structural models of the states were resolved by fFCS, MFD and hpFRET.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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