Abstract
Interactions of ribonucleic acids (RNA) with basic ligands such as proteins or aminoglycosides play a key role in fundamental biological processes. Native top-down mass spectrometry (MS) has recently been extended to binding site mapping of RNA–ligand interactions by collisionally activated dissociation, without the need for laborious sample preparation procedures. The technique relies on the preservation of noncovalent interactions at energies that are sufficiently high to cause RNA backbone cleavage. In this study, we address the question of how many and what types of noncovalent interactions allow for binding site mapping by top-down MS. We show that proton transfer from protonated ligand to deprotonated RNA within salt bridges initiates loss of the ligand, but that proton transfer becomes energetically unfavorable in the presence of additional hydrogen bonds such that the noncovalent interactions remain stronger than the covalent RNA backbone bonds.
Highlights
Interactions of ribonucleic acids (RNA) with basic ligands such as proteins or aminoglycosides play a key role in fundamental biological processes
We show that proton transfer from protonated ligand to deprotonated RNA within salt bridges initiates loss of the ligand, but that proton transfer becomes energetically unfavorable in the presence of additional hydrogen bonds such that the noncovalent interactions remain stronger than the covalent RNA backbone bonds
Similar products and product yields were observed in collisionally activated dissociation (CAD) of (M + Tma − 4H)3− ions of RNA 1 (Figure S1b), the collision energy required for 50% fragment formation by breaking of covalent backbone bonds, E50(c), increased by ∼5% from 46.7 ± 0.1 to 48.8 ± 0.1 eV (Figure 1a,d)
Summary
Interactions of ribonucleic acids (RNA) with basic ligands such as proteins or aminoglycosides play a key role in fundamental biological processes. We have recently shown that the electrostatic interactions between TAR ribonucleic acid (RNA) and a peptide comprising the arginine-rich binding region of tat protein are sufficiently strong in the gas phase to survive RNA backbone bond cleavage by CAD, allowing its use for probing tat binding sites in TAR RNA.[10] X-ray crystallography[23] and solution NMR24 were so far unsuccessful in providing a detailed picture of the TAR−tat binding interface, but highly converging structures of TAR RNA in a complex with a cyclic tat peptide mimetic showed interactions of all basic residues with phosphodiester moieties[25] and excellent agreement with the binding site predicted from our MS data.[10] At the solution pH of 7.7 used in our study, the arginine (pK > 11)[26] and lysine (pK > 10.5)[26,27] side chains of tat peptide should be protonated and available for salt bridge (SB) formation with the deprotonated RNA phosphodiester moieties (pK 1−3).[28] We attributed the unusual strength of TAR−tat interactions in the gas phase to electrostatic interactions, of which salt bridges are thought to provide the highest contribution to stability.[20,29] the question remains as to how many and what types of interactions, alone or in combination, are sufficient for probing of RNA−ligand binding sites by CAD. Seven peptides (GR, VR, DR, ER, KR, RR, NGR) and the fixed-charge ligand tetramethylammonium (Tma) were investigated in 1:1 complexes with seven different RNAs (Table 1)
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