Pitfalls in measurements of R1 relaxation rates of protein backbone 15N nuclei
Abstract
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 800Â*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 800Â*MHz, when non-mono-exponential decay is involved, it is advisable to consider elimination of the shortest delays (typically up to 0.32Â*s) or bi-exponential fitting.
Source: Journal of Biomolecular NMR