Pitfalls in measurements of R₁ relaxation rates of protein backbone ¹⁵N nuclei

The dynamics of the backbone and side-chains of protein are routinely studied by interpreting experimentally determined ¹⁵N spin relaxation rates. R₁(¹⁵N), the longitudinal relaxation rate, reports on fast motions and encodes, together with the transverse relaxation R₂, structural information about the shape of the molecule and the orientation of the amide bond vectors in the internal diffusion frame. Determining error-free ¹⁵N longitudinal relaxation rates remains a challenge for small, disordered, and medium-sized proteins. Here, we show that mono-exponential fitting is sufficient, with no statistical preference for bi-exponential fitting up to 800 MHz. A detailed comparison of the TROSY and HSQC techniques at medium and high fields showed no statistically significant differences. The least error-prone DD/CSA interference removal technique is the selective inversion of amide signals while avoiding water resonance. The exchange of amide with solvent deuterons appears to affect the rate R₁ of solvent-exposed amides in all fields tested and in each DD/CSA interference removal technique in a statistically significant manner. In summary, the most accurate R₁(¹⁵N) rates in proteins are achieved by selective amide inversion, without the addition of D₂O. Importantly, at high magnetic fields stronger than 800 MHz, when non-mono-exponential decay is involved, it is advisable to consider elimination of the shortest delays (typically up to 0.32 s) or bi-exponential fitting.