Advances in the use of FRET for protein structure determination and its application to the study of the spindle pole body.
Eric Muller, Brian Snydsman, Bryan Sundin, Bethany Fox, Dale Hailey, Trisha Davis
Dept. Biochemistry, Box 357350, University of Washington, 1959 NE Pacific St., Seattle, WA, 98195, US
Fluorescence Resonance Energy Transfer, or FRET, is a valuable tool in the study of large macromolecular complexes. FRET complements both cryo-EM tomography and single particle cryo-EM since FRET measurements can position proteins within the overall conformations determined by EM. FRET is particularly well suited to examine dynamic conformational changes, since the extent of energy transfer is exquisitely sensitive to small differences in distance. To date the greatest success of applying FRET has occurred in vitro, under conditions in which the investigator has greatest control over the choice of donor and acceptor fluorescent tags. We have developed methodologies and standards that allow the use of CFP and YFP in live cell FRET experiments. With the spindle pole body (SPB) as our model, we will show in detail the advantage of our new metric for measuring FRET. In addition the geometry of the SPB has permitted a correlation between our FRET metric and protein to protein distances. By incorporating distance constraints derived from FRET measurements into symmetry constraints imposed by cryo-EM analysis, a model of the SPB was created. The model provides new insights into the assembly and organization of the SPB.
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