Journal
JOURNAL OF BIOMOLECULAR NMR
Volume 53, Issue 3, Pages 209-221Publisher
SPRINGER
DOI: 10.1007/s10858-012-9626-5
Keywords
Backbone dynamics; Relaxation; TROSY; Perdeuterated proteins; Cross-correlated relaxation; Water saturation
Categories
Funding
- Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH)
- Intramural AIDS-Targeted Antiviral Program of the Office of the Director, NIH
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While extracting dynamics parameters from backbone N-15 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 N-15 R-1 and R-2 or R-1 rho relaxation rates as well as heteronuclear N-15-{H-1} 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 N-15 magnetization through the NMR-sensitive H-1 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 R-2'/R-1' 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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