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Title of Journal: J Biomol NMR

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Abbravation: Journal of Biomolecular NMR

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Springer Netherlands

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DOI

10.1007/bf00581865

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1573-5001

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A rigid disulfidelinked nitroxide side chain simp

Authors: Nicolas L Fawzi Mark R Fleissner Nicholas J Anthis Tamás Kálai Kálmán Hideg Wayne L Hubbell G Marius Clore
Publish Date: 2011/09/27
Volume: 51, Issue: 1-2, Pages: 105-
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Abstract

The measurement of 1H transverse paramagnetic relaxation enhancement PRE has been used in biomolecular systems to determine longrange distance restraints and to visualize sparselypopulated transient states The intrinsic flexibility of most nitroxide and metalchelating paramagnetic spinlabels however complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center Here we present a novel disulfidelinked nitroxide spin label R1p as an alternative to these flexible labels for PRE studies When introduced at solventexposed αhelical positions in two model proteins calmodulin CaM and T4 lysozyme T4L EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label generated by reacting cysteine with the spin labeling compound often referred to as MTSL Further only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1 Together these observations suggest that the nitroxide adopts a single fixed position when R1p is placed at solventexposed αhelical positions greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin labelWe thank Vincenzo Venditti and Mengli Cai for helpful suggestions Dusty Baber and Jinfa Ying for assistance with NMR spectrometers and Evan Brooks and Mária Balog for expert technical assistance This work was in part supported by funds from the Intramural Program of the NIH NIDDK and the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the NIH to GMC NIH grants R01EY05216 to WLH and the Jules Stein Professorship Endowment to WLH The synthesis of new spin label reagents was supported by Hungarian National Research Funds OTKA K81123

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