SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Accessibility and Dynamics Of Nitroxide Side Chains In T4 Lysozyme Measured By Saturation Recovery EPR
Janusz Pyka 1, Jan Ilnicki 1, Christian Altenbach 2, Wayne L. Hubbell 2* and Wojciech Froncisz 1
1 Jagiellonian University, Poland
2 University of California, Los Angeles
* To whom correspondence should be addressed. E-mail: hubbellw{at}jsei.ucla.edu.
Submitted on January 5, 2005
Revised on March 9, 2005
Accepted on 25 May 2005
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Abstract |
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Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin labeled side chains placed along a regular helix extending from 128-135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin lattice relaxation rates of the nitroxides, which depend on the nitroxide side chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O2 or NiEDDA, global analysis of the saturation recovery provides the spin lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin lattice relaxation rates and the exchange rates along the 128-135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the time scale of spin lattice relaxation.
Key Words:
SDSL, electron spin-lattice relaxation
