Imino proton resonances

The well-resolved imino proton resonances from base-paired uracil and guanine resonances serve as powerful probes to characterize RNA-ligand interaction by NMR. The chemical shift of imino proton resonances is responsive to perturbations in the local chemical environment that result from RNA conformational... 

Figure 5.4. Titration of A-site oligonucleotide with paromomycin. Titration of the A-site oligonucleotide with paromomycin yields a specific 1 1 complex. Imino proton resonances for U1490 and G1491, which shift significantly upon binding of paromomycin, are labeled. The intermediate point in the titration demonstrates the appearance of new peaks from the bound form of the RNA.

Observation of the imino-proton resonances - albeit somewhat shifted and broadening at low temperatures - of the central G C base pairs, indicates that hydrogen bonding (Watson-Crick type or related) is still possible after platination. 

Figure 19.3 Effect of the addition of on the imino proton resonance signals at 4 °C of the duplex without a single-stranded end (14mer) (a) and the duplex with an ACCA 3 -terminus at base pair 1 (18mer) (b). In each case, the lower trace belongs to the solution without Mn " and the upper trace gives the spectrum after addition of either 7.5 pM a) or 5 pM b) MnCl2. The solutions contain 100 mM NaCl and either 7.5 mM a) or 5 mM b) MgCl2.

Figure 19.14 The imino proton resonances in the NMR spectrum of free Phe-tRNA and in ternary complex with EF-Tu GTP and EF-Tu GDP [47]. a) 270 iM tRNA, b)270 pM Phe-tRNA EF-Tu GTP ternary complex, c) the same complex as in (b) after GTP hydrolysis and deacylation of Phe-tRNA . The dashed lines in (b) and (c) correspond to the NMR imino proton spectrum of free tRNA , as shown in (a). The numbers denote the imino proton resonances of the base pairs in the tRNA acceptor stem as assigned by [74]. The numbers in circles indicate the signals of the first three imino protons in the acceptor stem, which disappear from the NMR spectrum after ternary complex formation.

Figure 5-51 (A) The low-field region of the one-dimensional H NMR spectrum of E. coli tRNAjVal at 27°C in H20. Resonances are identified by letters A - X. (B) NOESY spectrum of the same tRNA under similar conditions showing the imino-imino NOEs. In the lower right sector the connectivity traces of the acceptor helix and dihydrouridine helix are shown as solid and dotted lines, respectively. In the NOESY sample the two protons in peak EF are partially resolved whereas the two protons in peak T have coalesced. (C) NOESY spectrum of E. coli tRNA,Val at 32°C showing the imino and aromatic proton regions. AU-type imino protons have been connected horizontally by a dotted line to the cross-peak of their proximal C2-H or C8-H in the 7 to 9 ppm region, which has been labeled with the corresponding lower-case letter. From Hare et al.669 Courtesy of Brian Reid.

Replacement of the proton at N1 of G by a metal ion has qualitatively similar consequences as substitution of the imino proton of T and U. The Pt(II) binding to this site increases the basicity of the purine ring, but it is not clear how the residual electron density is distributed. The N1 platinated guanine bases titrate with a pA a of 5 (176, 177), and from the high pD dependence of the H8 resonances in the H NMR spectra it is concluded that the most likely protonation site is N7 (cf. Fig. 16). In its protonated state, the nucleobase represents again a metal-stabilized rare tautomer. 

Figure 24 Portion of a 500-MHz 2-D NOESY spectrum in 90% H20/10% D2O, 10 °C, of the hairpin rihozyme B domain in the presence of 1 mM cobalt hexamine, which resonates at 3.65 ppm. NOEs could he unambiguously assigned to the imino protons of nucleotides G2, U3, and G4 (a) and the amino protons of C35 and C37 and the H8 protons of G2 and G7 (h). NOEs with imino (a) and amino (b) protons are shown. (Adapted from Butcher, Allain and Feigon. 2000 American Chemical Society)...

In the next step, because of the basicity of the medium and the acidic protons that are present, the amino proton is removed prior to nucleophilic substitution of the second ester carbonyl. Because of the resonance stabilization possible for the resulting anion, the amino proton, not the imino proton, reacts. Removal of a proton prior to cyclization also eliminates the need for the last step shown in the problem. 

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