EBS1*
Up to this stage, two metal ions were defined to bind at specific regions in the loop. However, it is not clear yet, if the metal ions stick to these sites. Since the loop shows a certain flexibility, the metal ions, which are bound to the loop, might be also influenced by the dynamic behaviour of the loop. Thus, the calculation of the metal ion binding sites in the loop was repeated with newly defined binding sites (Figure 92). The first binding site in the loop L1b now includes the nucleotides A10, U11 and U12. At these nucleotides a binding site was previously determined by Tb3+ cleavage assays.(97) The second binding site in the loop L2b is located at the end of the loop, involving A16 to G21.
The affinity constants were calculated with and without the incorporation of the equilibrium concentrations of free and bound metal ions, i.e. with ISTARv2.2 and ISTARv2.3, respectively. The results for all four binding sites in d3'-EBS1* with the newly defined binding sites in the loop are listed in Table 19. After the first round of calculation, in which the total amount of [Mg2+] is considered for every binding site, the log KA,av1 values for
the 5'-end and the helix 1 are the same as in the previous calculation (Table 17 and 19). This is of course not surprising as these binding sites are defined equally in both calculations. However, the final log KA,fin values are slightly increased, i.e. with the newly defined binding
sites, the stability of the Mg2+ complex of the 5'-end is increased by 0.08 ± 0.05 log units with the classical ISTARv2.2 version and by 0.02 ± 0.01 log units with ISTARv2.3, which includes the equilibrium concentrations. The final log KA,fin value of helix 1 experiences an
increase of 0.03 ± 0.01 log units with ISTARv2.2 and with ISTARv2.3 it is the same within the error limits.
Comparing the log KA,av1 values of the binding sites loop 1b and loop 2b after the first
iteration round, it is already obvious that the binding site loop 1b with a log KA,av1 of 2.31 ±
0.05 mM–1 has now a higher affinity to Mg2+ than loop 2b with 2.02 ± 0.05 mM–1. The log
KA,fin of loop 1b gives a value of 2.71 ± 0.01 mM–1 with ISTARv2.2 and 2.63 ± 0.03 mM–1
with ISTARv2.3, whereas loop 2b has a log KA,fin value of 2.47 ± 0.02 mM–1 if calcultated
Figure 92 Secondary structure of d3'-EBS1*. The two
metal ion binding sites in the loop were rearranged and the calculation repeated. The binding sites are coloured in blue (5'-end), green (helix 1), red (loop 1b) and orange (loop 2b).
without the equilibrium concentrations of bound and unbound M2+, and 2.38 ± 0.02 mM–1 including the equilibrium, respectively. These results can be traced back to the individual log
KA,est values of the single protons in the loop region (see Appendix 10A). Since the individual
log KA,est values contribute to the final affinity constants for each binding site, these values are
highly dependent on the individual log KA,est values. A20H2, A20H8 and G19H8 have log
KA,est values below 2, thus the final log KA,fin values for the single binding sites depend on the
incorporation of these protons into the site. However, besides these three relatively low log
KA,est values, all other values of the individual protons in the loop are roughly in the same
range. Thus, it can be generally accepted that the two metal ions bind with the same affinity to the loop and that these metal ions are somehow spread over the loop to cover a large range. Having a look at the solution structure of d3'-EBS1*, the first suggestion of the binding sites in the loop as shown in Figure 89 is the more likely one. This assumption is based on the arrangement of the nucleotides in the loop. A10, U11, U12, A20 and G21 are located such that one Mg2+ ion can cover this range, whereas the nucleotides at the 3'-end of the loop, A16 to G21, span a relatively large range which cannot be covered by only one metal ion. Thus, it is assumed that the binding sites shown in Figure 89 are the more reliable ones.
Table 19 Affinity values log KA for Mg2+ binding to d3'-EBS1* in D2O with newly defined binding sites in the
loop (see Figure 92). Listed are the averaged log KA values (1σ) at the four high affinity binding sites, obtained
from the change in chemical shifts of all aromatic and H1' protons after various rounds of iterative corrections
for the Mg2+ concentration that is available at a certain site. The log KA values are shown for the calculation with
ISTARv2.2 (non-shaded lines) and the modified version of ISTARv2.3 (see text), which takes the equilibrium
concentrations of the free and bound M2+ species in solution into account (shaded lines).
Calculation methoda Binding site log KA,av1 [mM–1] log KA,av2 [mM–1] log KA,av3 [mM–1] log KA,av4 [mM–1] log KA,av5 [mM–1] log KA,fin b [mM–1] ∆fin – av1 c [mM–1] ISTARv2.2 5'-end 3.30 ± 0.13 3.50 ± 0.14 3.68 ± 0.16 3.75 ± 0.18 3.79 ± 0.19 3.84 ± 0.06 0.54 ± 0.13 ISTARv2.3 5'-end 3.30 ± 0.13 3.41 ± 0.13 3.48 ± 0.14 3.49 ± 0.14 3.49 ± 0.14 3.50 ± 0.01 0.20 ± 0.13 ISTARv2.2 Helix 1 (H1) 2.38 ± 0.04 2.64 ± 0.02 2.74 ± 0.03 2.78 ± 0.03 2.79 ± 0.03 2.80 ± 0.01 0.42 ± 0.04 ISTARv2.3 Helix 1 (H1) 2.38 ± 0.04 2.60 ± 0.03 2.65 ± 0.03 2.66 ± 0.03 2.66 ± 0.03 2.66 ± 0.01 0.28 ± 0.04 ISTARv2.2 Loop 1(L1b) 2.31 ± 0.05 2.60 ± 0.03 2.66 ± 0.02 2.69 ± 0.02 2.71 ± 0.02 2.71 ± 0.01 0.40 ± 0.05 ISTARv2.3 Loop 1(L1b) 2.31 ± 0.05 2.56 ± 0.03 2.61 ± 0.03 2.62 ± 0.03 2.63 ± 0.03 2.63 ± 0.03 0.32 ± 0.05 ISTARv2.2 Loop 2 (L2b) 2.02 ± 0.05 2.34 ± 0.03 2.41 ± 0.02 2.45 ± 0.02 2.47± 0.02 2.47 ± 0.02 0.45 ± 0.05 ISTARv2.3 Loop 2 (L2b) 2.02 ± 0.05 2.32 ± 0.04 2.37 ± 0.03 2.38 ± 0.03 2.38 ± 0.03 2.38 ± 0.02 0.36 ± 0.05 The chemical shift changes were obtained from 2D [1H,1H]-NOESY spectra of a 0.5 mM d3'-EBS1* RNA at pD 6.96 in 10 mM KCl at 25 °C. All error limits correspond to one standard deviation (1σ). aIn the modified ISTAR version an additional variable v is defined, taking the
equilibrium between the bound and unbound state of the metal ion into account (see text). bThe maximal log K
A,fin values correspond to the
limiting value of an asymptotic fit, which was obtained by plotting log Ka values after each iteration round versus the number of the iteration
rounds. The errors from the fits were multiplied by three to obtain reasonable error limits. cDifference in log K
A between log KA,av1 and the
Results and Discussion 127