Amide proton relaxation measurements employing a highly deuterated protein

Abstract

Proton NMR longitudinal and transverse relaxation rates of unlabelled proteins are generally dominated by the many 1H– 1H dipolar interactions so that spin diffusion, rather than molecular or internal motions, governs longitudinal relaxation. Here, relaxation measurements of backbone amide proton ( 1H N) magnetisations have been carried out employing the 99% 2H, 98% 15N labelled, small 2F2 protein domain in 10%/90% H 2O/D 2O solution. Under these conditions, the longitudinal relaxation rates exhibit time constants, T 1 *=1/ R 1 * if described by a mono-exponential, within the range of 3.0 to 18.7 s—a wide range which indicates that the phenomenon of spin diffusion has been greatly reduced. The majority of 1H N nuclei in this sample (pH 4.0 and 5 °C) exhibit chemical exchange with solvent that couples their longitudinal relaxation to that of the solvent. For the subset of 1H N nuclei not undergoing detectable solvent chemical exchange, the R 1 * rates correlate well with their individual 1H N,O/ 2H N,O structural environments. The correlation for corresponding transverse relaxation rates, R 2 * was found to be less good. Longitudinal relaxation measurements in 1%/99% H 2O/D 2O solution identify a further subset of 1H N nuclei which exhibit essentially indistinguishable R 1 * rates in both 1% and 10% H 2O, implying that averaging of rates from spin diffusion processes and different 2F2 isotopomer populations are negligible for these 1H N sites. In addition to a high sensitivity to structural parameters, model calculations predict 1H N relaxation rates to exhibit pronounced sensitivity to internal dynamics.