Native Top-Down Mass Spectrometry of TAR RNA in Complexes with a Wild-Type tat Peptide for Binding Site Mapping.

Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures. The characterization of RNA-protein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, or strategies based on chemical crosslinking, however, can be quite challenging. Herein, we have explored the use of an alternative method, native top-down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single-residue level of RNA. Our data show that the electrostatic interactions between HIV-1 TAR RNA and a peptide comprising the arginine-rich binding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNA by collisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TAR RNA by top-down MS. Moreover, the MS data reveal time-dependent 1:2 and 1:1 stoichiometries of the TAR-tat complexes and suggest structural rearrangements of TAR RNA induced by binding of tat peptide.

Native mass spectrometry reveals the initial binding events of HIV-1 rev to RRE stem II RNA.

Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection.

Replacing H+ by Na+ or K+ in phosphopeptide anions and cations prevents electron capture dissociation.

By successively replacing H+ by Na+ or K+ in phosphopeptide anions and cations, we show that the efficiency of fragmentation into c and z˙ or c˙ and z fragments from N-Cα backbone bond cleavage by negative ion electron capture dissociation (niECD) and electron capture dissociation (ECD) substantially decreases with increasing number of alkali ions attached. In proton-deficient phosphopeptide ions with a net charge of 2-, we observed an exponential decrease in electron capture efficiency with increasing number of Na+ or K+ ions attached, suggesting that electrons are preferentially captured at protonated sites. In proton-abundant phosphopeptide ions with a net charge of 3+, the electron capture efficiency was not affected by replacing up to four H+ ions with Na+ or K+ ions, but the yield of c, z˙ and c˙, z fragments from N-Cα backbone bond cleavage generally decreased next to Na+ or K+ binding sites. We interpret the site-specific decrease in fragmentation efficiency as Na+ or K+ binding to backbone amide oxygen in competition with interactions of protonated sites that would otherwise lead to backbone cleavage into c, z˙ or c˙, z fragments. Our findings seriously challenge the hypothesis that the positive charge responsible for ECD into c, z˙ or c˙, z fragments can generally be a sodium or other metal ion instead of a proton.

Interactions of Protonated Guanidine and Guanidine Derivatives with Multiply Deprotonated RNA Probed by Electrospray Ionization and Collisionally Activated Dissociation.

Interactions of ribonucleic acid (RNA) with guanidine and guanidine derivatives are important features in RNA-protein and RNA-drug binding. Here we have investigated noncovalently bound complexes of an 8-nucleotide RNA and six different ligands, all of which have a guanidinium moiety, by using electrospray ionization (ESI) and collisionally activated dissociation (CAD) mass spectrometry (MS). The order of complex stability correlated almost linearly with the number of ligand atoms that can potentially be involved in hydrogen-bond or salt-bridge interactions with the RNA, but not with the proton affinity of the ligands. However, ligand dissociation of the complex ions in CAD was generally accompanied by proton transfer from ligand to RNA, which indicated conversion of salt-bridge into hydrogen-bond interactions. The relative stabilities and dissociation pathways of [RNA+m L-n H] n- complexes with different stoichiometries (m=1-5) and net charge (n= 2-5) revealed both specific and unspecific ligand binding to the RNA.

Unfolding and Folding of the Three-Helix Bundle Protein KIX in the Absence of Solvent.

Electron capture dissociation was used to probe the structure, unfolding, and folding of KIX ions in the gas phase. At energies for vibrational activation that were sufficiently high to cause loss of small molecules such as NH3 and H2O by breaking of covalent bonds in about 5% of the KIX (M + nH)(n+) ions with n = 7-9, only partial unfolding was observed, consistent with our previous hypothesis that salt bridges play an important role in stabilizing the native solution fold after transfer into the gas phase. Folding of the partially unfolded ions on a timescale of up to 10 s was observed only for (M + nH)(n+) ions with n = 9, but not n = 7 and n = 8, which we attribute to differences in the distribution of charges within the (M + nH)(n+) ions. Graphical Abstract ᅟ.