Impact of spin label rigidity on extent and accuracy of distance information from PRE data

Paramagnetic relaxation enhancement (PRE) is a versatile tool for NMR spectroscopic structural and kinetic studies in biological macromolecules. Here, we compare the quality of PRE data derived from two spin labels with markedly different dynamic properties for large RNAs using the I-A riboswitch aptamer domain (78 nt) from Mesoplamsa florum as model system. We designed two I-A aptamer constructs that were spin-labeled by noncovalent hybridization of short spin-labeled oligomer fragments. As an example of a flexible spin label, U-Ureido-TEMPO was incorporated into the 3' terminal end of helix P1 while, the recently developed rigid spin-label Cm was incorporated in the 5' terminal end of helix P1. We determined PRE rates obtained from aromatic C-13 bound proton intensities and compared these rates to PREs derived from imino proton intensities in this sizeable RNA (similar to 78 nt). PRE restraints derived from both imino and aromatic protons yielded similar data quality, and hence can both be reliably used for PRE determination. For NMR, the data quality derived from the rigid spin label Cm is slightly better than the data quality for the flexible (TEMPO)-T-Ureido as judged by comparison of the structural agreement with the I-A aptamer crystal structure (3SKI).