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Measurement of 15N relaxation rates in perdeuterated proteins by TROSY-based methods

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Abstract

While extracting dynamics parameters from backbone 15N relaxation measurements in proteins has become routine over the past two decades, it is increasingly recognized that accurate quantitative analysis can remain limited by the potential presence of systematic errors associated with the measurement of 15N R1 and R2 or R1ρ relaxation rates as well as heteronuclear 15N-{1H} NOE values. We show that systematic errors in such measurements can be far larger than the statistical error derived from either the observed signal-to-noise ratio, or from the reproducibility of the measurement. Unless special precautions are taken, the problem of systematic errors is shown to be particularly acute in perdeuterated systems, and even more so when TROSY instead of HSQC elements are used to read out the 15N magnetization through the NMR-sensitive 1H nucleus. A discussion of the most common sources of systematic errors is presented, as well as TROSY-based pulse schemes that appear free of systematic errors to the level of <1 %. Application to the small perdeuterated protein GB3, which yields exceptionally high S/N and therefore is an ideal test molecule for detection of systematic errors, yields relaxation rates that show considerably less residue by residue variation than previous measurements. Measured R2′/R1′ ratios fit an axially symmetric diffusion tensor with a Pearson’s correlation coefficient of 0.97, comparable to fits obtained for backbone amide RDCs to the Saupe matrix.

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Acknowledgments

We thank Dennis A Torchia and Alex Grishaev for helpful discussions and computer simulations, Frank Delaglio for assistance with the evaluation of relaxation data and Alex Maltsev for help with the expression and purification of GB3. This work was funded by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH) and the Intramural AIDS-Targeted Antiviral Program of the Office of the Director, NIH.

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  1. Laboratory of Chemical Physics (LCP), DHHS NIDDK, National Institutes of Health, Building 5, Room 126, 9000 Rockville Pike, Bethesda, MD, 20892-0520, USA

    Nils-Alexander Lakomek, Jinfa Ying & Ad Bax

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  1. Nils-Alexander Lakomek
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Correspondence to Ad Bax.

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Lakomek, NA., Ying, J. & Bax, A. Measurement of 15N relaxation rates in perdeuterated proteins by TROSY-based methods. J Biomol NMR 53, 209–221 (2012). https://doi.org/10.1007/s10858-012-9626-5

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