Distance measurements by fluorescence energy homotransfer.
Evaluation in T4 Lysozyme and correlation with dipolar
coupling between spin labels
Ping Zou 1, Kavitha Surendhran 1 and Hassane Mchaourab 1*
1 Vanderbilt University
* To whom correspondence should be addressed. E-mail: hassane.mchaourab{at}vanderbilt.edu.
Submitted on October 11, 2006
Revised on October 26, 2006
Accepted on 14 November 2006
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Abstract |
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We demonstrate the feasibility and practical limitations of using steady state anisotropy to determine distances from fluorescence homotransfer in the context of a protein of known crystal structure. Eight double mutants of T4 lysozyme spanning the distance range between 20-50 Å were labeled with an MTS derivative of fluorescein. The measured distances in liquid solution are in agreement with those determined from dipolar coupling between spin labels in the frozen state. They can be interpreted in the context of the crystal structure after accounting for the probe linking arm. Overall, the results establish the necessary calibration for this spectroscopic ruler. The measurement of similar distance trends using independent probes sets the stage for the complementary use of homotransfer and dipolar coupling in the determination of static structures and detection of conformational changes.
Key Words:
EPR, dipolar spectroscopy, fluorescence, homotransfer, spin labels
