Fig. 2: Verifying a mutant mimic of the HIV-1 TAR ES2 using chemical shift fingerprinting.

a The TAR GS exists in dynamic equilibrium with a sparsely populated and short-lived excited conformational state, ES2. The secondary structure of ES2 was deduced based on prior ¹³C and ¹⁵N chemical shifts^21,23 obtained using NMR R_1ρ experiments for residues throughout the lower helix, bulge, upper stem, and apical loop (shown in color). Residues showing exchange contributions in the ¹H CEST experiment are circled in pink. b The TAR^ES2 mutant^10,22,23 stabilizes an ES2-like conformation as the dominant GS. c The base pairs and mismatches in ES2 which were verified using the ¹H CEST experiments. d Correlation plot comparing the difference between the ¹H, ¹³C, and ¹⁵N chemical shifts measured between the TAR ES2 and GS (Δω_(ES2-GS) = ω_ES2 - ω_GS) using NMR R_1ρ (¹³C and ¹⁵N) and CEST (¹H) with the corresponding difference in chemical shifts (Δω_(mutant-wt) = ω_mutant – ω_wt) obtained from comparing the GS chemical shifts for TAR^ES2 and wtTAR. For resonances belonging to G33 near the site of mutation (in open symbols), values were derived from the G28U TAR ES2 mutant²¹. Δω are color-coded according to the structural elements in (a). The N represents the total number of NMR probes used for the comparison in the correlation plots. e Representative ¹H CEST profile for U38-H3 showing an exchange contribution. RF field powers used are color-coded. Remaining data are shown in Supplementary Fig. 1a. f Comparison of the population (p_ES2) and kinetic exchange rate (k_ex = k₁ + k_-1) obtained by ¹H CEST with values measured previously²¹ using ¹³C, ¹⁵N R_1ρ. R_1ρ or CEST measurement data presented here are the mean values ± 1 s.d. from Monte Carlo simulations (number of iterations = 500) as described in Methods. The errors in exchange parameters derived from ¹H CEST were set equal to the fitting errors determined as the square root of the diagonal elements of the covariance matrix. g Comparing the difference between the ¹H chemical shifts measured between the TAR ES2 and GS (Δω = ω_ES2 - ω_GS) using ¹H CEST (in red) with the corresponding difference in chemical shifts (Δω = ω_mutant – ω_wt) obtained from comparing the GS chemical shifts for the mutant TAR^ES2 and wtTAR (in blue). Error bars denote the error of exchange parameters.