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

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.

Probing the transient dark state of substrate binding to GroEL by relaxation-based solution NMR

“…S5) (21), 15 N-R 1ρ (Fig. S6) (38), 15 N-DEST (Fig. S7) (21), and 15 N-CPMG (single quantum) relaxation dispersion (39) experiments were carried out as described previously.…”

A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR

“…We used an array of TROSY-based triple-resonance assignment experiments (HNCO, HN(CA) CO A full set of R 1 , R 1 , and 15 N-{ 1 H} NOE relaxation spectra were recorded at 600 MHz and supplemented with R 1 measurements at 900 MHz from a 0.9 mM perdeuterated, amide 1 H sample using TROSY readout methods (30). R 2 rates were obtained from rotating frame R 1 rates (31) measured under a spin-lock field strength of 2 kHz, after correction for the 15 N off-resonance, tilted field.…”

Internal Dynamics of the Homotrimeric HIV‐1 Viral Coat Protein gp41 on Multiple Time Scales

“…1 H} NOE and transverse 15 N chemical-shift anisotropy (CSA)-dipolar cross-correlated relaxation rates, h xy , were measured at both 600 and 800 MHz by TROSY-based methods optimized for perdeuterated proteins [19] ( Figure 3 a-d). Limited sensitivity did not permit the use of 3D methods, [20] and only data for amides that could be resolved in the 2D 15 N-1 H TROSY-HSQC spectrum are shown in Figure 3.…”