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

Abstract

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-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 aromaticC 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 TEMPO as judged by comparison of the structural agreement with the I-A aptamer crystal structure (3SKI).

Accurate Electron–Nucleus Distances from Paramagnetic Relaxation Enhancements

“…In the case of nitroxide tags, flexibility of the tether between nitroxide group and protein backbone poses an intrinsic difficulty to translate PREs into distances between the nitroxide and the nuclear spins. Where PREs generated by nitroxide tags have been calibrated against electron–nucleus distances, the uncertainties were large and distances outside the range 15–24 Å proved inaccessible in solution. ,, Similar results have also been obtained for RNA. − In the example of a copper protein, PREs determined from the longitudinal relaxation rates of protons, R 1 ( 1 H), correlated reasonably well with Cu 2+ – 1 H distances measured in a solution NMR structure, but with systematic deviations for distances greater than 10 Å . For perdeuterated proteins in the solid state, distance measurements up to 32 Å have been reported, but with very large outliers…”

A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full‐Length TAR RNA and Investigation by In‐Line Probing and EPR Spectroscopy

“…Apart from pure EPR studies, long‐range distances derived from PELDOR experiments can provide valuable complementary data for structure determination by NMR spectroscopy . Spin‐labeled RNA samples are also required for NMR studies using paramagnetic relaxation enhancement . Although, compared to PELDOR, 5–10 times larger sample quantities are required, upscaling of the methods described herein would not be infeasible.…”

“…[30,31] Spin-labeled RNA samples are also required for NMR studies using paramagnetic relaxation enhancement. [76] Although, compared to PELDOR, 5-10 times larger sample quantities are required, upscaling of the methods described herein would not be infeasible.…”

Pseudo aptamer expands aptamer’s applications

“…The effect often attributes to both non-specific and specific interactions, since the recognition between nucleotide acid and proteins involves side chains and backbone interactions in the manner of the nucleic acid bases amino acid codes, 47,48 hydrogen bonds, [49][50][51][52][53] charge-charge interactions, [54][55][56][57][58][59] hydrophobicity or hydrophilicity 60,61 conformations, 62-69 rigidity etc. [70][71][72] Among the modifications on backbone, sulfurization is one of common ways. In doing so, dithiazol-one based derivatives as the most efficient sulfurization reagents have been employed to introduce sulfur element.…”