Metal Ion–RNA Interactions Studied via Multinuclear NMR

Abstract

Metal ions are indispensable for ribonucleic acids (RNAs) folding and activity. Firstly they act as charge neutralization agents, allowing the RNA molecule to attain the complex active three dimensional structure. Secondly, metal ions are eventually directly involved in function.Nuclear magnetic resonance (NMR) spectroscopy offers several ways to study the RNAmetal ion interactions at an atomic level. Here we first focus on special requirements for NMR sample preparation for this kind of experiments: the practical aspects of in vitro transcription and purification of small (< 50 nt) RNA fragments is described, as well as the precautions that must be taken into account when a sample for metal ion titration experiments is prepared. Subsequently, we discuss the NMR techniques to accurately locate and characterize metal ion binding sites in a large RNA. For example, 2 J-[ 1 H, 15 N]-HSQC (Heteronuclear Single Quantum Coherence) experiments are described to qualitatively distinguish between different modes of interaction. Finally, part of this last section is devoted to data analysis, this is how to calculate intrinsic affinity constants.

On the Role of NMR Spectroscopy for Characterization of Antimicrobial Peptides

“…In the case of recombinant-expressed peptides, weak alignment of peptides using acrylamide gels has been used to obtain residual dipolar couplings for orientational-dependent restraints (151–153). The positioning of peptides with respect to micelle surfaces has been inferred by paramagnetic relaxation enhancement mapping (34, 38, 147, 154, 155), water exposure measurements based on exchange peaks with the water signal (156–158), detection of NOEs between protonated micelles and peptides (159–162), or saturation transfer techniques (163–165). …”

Covalent and non-covalent binding of metal complexes to RNA

“…Tb(III)) are currently used in RNA footprinting experiments to map secondary and tertiary structure as well as to identify metal ion binding sites [58,59]. Also, different transition metal ions, like Cd(II) and Mn(II), as well as cobalt(III)hexammine are used as Mg(II) mimics in NMR studies [60][61][62][63][64][65]. These aspects of metal ion-RNA interactions go beyond the scope of this review and will not be further discussed.…”

NMR Spectroscopy in Bioinorganic Chemistry

“…[30] (D) NMR solution structure of D6-27 with five Mg 2+ ions at their proposed sites shown as red spheres (PDB ID 2AHT) [6] prepared with MOLMOL. [20] Panels (A) and (C) are adapted from refs [14,30]. Panel (B) is reprinted from ref.…”

“…However, our recent results clearly reveal that even if it is not possible to directly observe the coordinating metal ions by NMR, indirect methods can still give valuable information that is important for a better understanding of these systems. [4,12,14,48] …”

“…Nuclear magnetic resonance (NMR) spectroscopy is a crucial technique that we use not only to obtain structural information on our molecules at an atomic level [6][7][8][9][10][11] but, even more important, to elucidate in detail their metal ion binding. [2,9,10,[12][13][14][15][16][17][18] Fig. 1 shows three examples of recently solved structures, belonging to the three families of biomolecules we are working with: the exon/intron binding site 1 (EBS1/IBS1; PDB ID 2K65) [19] of the yeast mitochondrial group II intron Sc.ai5γ, a silver modified duplex DNA (PDB ID 2KE8), [8] and the C-terminal domain of the seed-specific wheat MT Zn 6 E c -1, denoted as Zn 4 β E -E c -1 (PDB ID 2KAK).…”