Figure 3.

Mg²⁺-induced folding monitored by imino proton NMR. Spectra of 0.4 mM of P5abc in 10 mM sodium phosphate and 0.05 mM EDTA (pH 6.4) were measured at 20°C as a function of added MgCl₂. The total concentration of Mg²⁺ was 0 mM (a), 10 mM (b), 20 mM (c), and 30 mM (d). Solid and dashed lines represent the NOE connectivities as determined by 2D NOESY of 3 bp in stem P5b (G141⋅U162, U142⋅A161, and C143⋅G160) for the unfolded and folded conformations, respectively. With the addition of Mg²⁺, the four imino proton peaks of these base pairs decrease, and a new set of four imino proton peaks with the same NOE connectivities appear at different chemical shifts. This response is characteristic of a slow exchange between the folded and unfolded conformations. The percentages of folding estimated from the relative intensity of U142 in the folded and unfolded states are ≈50% at 20 mM Mg²⁺ and 80% at 30 mM Mg²⁺. Six imino proton peaks of three G⋅U pairs (G188⋅U135, G164⋅U177, and G174⋅U167) disappear during the folding; the peaks are indicated by arrows. NOESY spectra of c and d show that these six bases no longer form G⋅U pairs in the folded state. An asterisk indicates the sheared G⋅A base pair of tetraloop L5c that disappears on folding.