Paramagnetic-iterative relaxation matrix approach: extracting PRE-restraints from NOESY spectra for 3D structure elucidation of biomolecules.

Paramagnetic relaxation enhancement (PRE) can be used to determine long-range distance restraints in biomolecules. The PREs are typically determined by analysis of intensity differences in HSQC experiments of paramagnetic and diamagnetic spin labels. However, this approach requires both isotope- and spin-labelling. Herein, we report a novel method to evaluate NOESY intensities in the presence of a paramagnetic moiety to determine PRE restraints. The advantage of our approach over HSQC-based approaches is the increased number of available signals without the need for isotope labelling. NOESY intensities affected by a paramagnetic center were evaluated during a structure calculation within the paramagnetic iterative relaxation matrix approach (P-IRMA). We applied P-IRMA to a 14-mer RNA with a known NMR solution structure, which allowed us to assess the quality of the PRE restraints. To this end, three different spin labels have been attached at different positions of the 14-mer to test the influence of flexibility on the structure calculation. Structural disturbances introduced by the spin label have been evaluated by chemical shift analysis. Furthermore, the impact of P-IRMA on the quality of the structure bundles were tested by intentionally leaving out available diamagnetic restraints. Our analyses show that P-IRMA is a powerful tool to refine RNA structures for systems that are insufficiently described by using only diamagnetic restraints.

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, UreidoU-TEMPO was incorporated into the 3' terminal end of helix P1 while, the recently developed rigid spin-label Çm was incorporated in the 5' terminal end of helix P1. We determined PRE rates obtained from aromatic 13C bound proton intensities and compared these rates to PREs derived from imino proton intensities in this sizeable RNA (~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 Çm is slightly better than the data quality for the flexible UreidoTEMPO as judged by comparison of the structural agreement with the I-A aptamer crystal structure (3SKI).