2560 Nucleic Acids Research, 2007, Vol. 35, No. 8 the movement of 15NH4þ ions from the binding site, which he inner site are 64.2 and 50.7 kJ mol 1 for O1I and O2I is combined with T1 relaxation. Dynamics of 15NH4þ ion movements, respectively. Calculations according to Eyring movement and T1 relaxation are accelerated with increas- theory indicate activation enthalpies ( Hz) and entropies ( Sz) of 60.7 kJ mol 1 and –53.8 J mol 1 K 1 for O1I and ing temperature (Table 3), which is reﬂected in the 48.3 kJ mol 1 and –95.8 J mol 1 K 1 for O2I. observed increase in the rate constant d . On the other 2 hand, the fast relaxation mechanism characterized by rate constant d1 does not appear to be signiﬁcantly inﬂuenced by increasing temperature. At the shortest mixing time of DISCUSSION 13 ms, however, the autocorrelation peak is already d[G4(T4G4)3] has been folded into unimolecular reduced considerably, which renders the d1 rate constants G-quadruplex in 130 mM aqueous solution of 15NH4Cl. less precisely deﬁned. The values for individual binding This 28-mer sequence with the four G4 repeats folds sites at four temperatures are essentially the same, within into G-quadruplex with four G-quartets. Our NMR data experimental error. At 313 K, the rate constants d1 and d2 on folded oligonucleotide in the presence of 15NH4þ ions could not be determined unambiguously. Very good are consistent with the 3D structure determined earlier agreement between the experimental data points and by Wang and Patel (1) in the presence of Naþ ions. Equation (3) was achieved for a range of d1 and d2 values, Three 15NH4þ ions have been localized within the with an appropriate adjustment of parameter f (Table 3). intramolecular d[G4(T4G4)3] quadruplex structure. In addition, cations have been shown to move among their Activation energy preferred binding sites within G-quadruplex and with bulk The temperature-dependent k values for the O1I and solution. Their movement has been analyzed in a O2I processes from Table 1 were used to construct an quantitative manner through the interpretation of several Arrhenius plot (Figure 7). The extracted activation cross-peaks in 2D 1H–15N NzExHSQC spectra. energies for exchange of 15NH4þ ions from the outer sites Cross-peaks in NzExHSQC spectra corresponding to t- 15NH4þ ions that have moved from one to the other o binding site are very weak relative to the autocorrelation t- peaks especially in comparison to bulk. Initially cross- peak volumes increase with lengthening of mixing time as more and more 15NH4þ ions move from their original locations. At certain mixing time ( m4200 ms), inﬂection point is observed when relaxation overtakes 15NH4þ ion movement, which results in decrease of cross-peak’s volume. Two well-resolved cross-peaks (O1I and O2I) correspond to the movement of 15NH4þ ions from the Figure 6. Relative volumes of autocorrelation peaks as a function of two outer binding sites into the inner binding site. For mixing time ( m) at 283 K (A) and 313 K (B). Black ﬁlled squares, blue these two cross-peaks, individual exchange rate constants open triangles and red open circles represent the experimental points and relaxation times have been calculated. At 283 K, for the autocorrelation peaks I, O1 and O2, respectively. Curves exchange rate constants for O1I and O2I processes are 0.05 represent the best ﬁts of the experimental data to Equation (3). and 0.07 s 1, respectively. The corresponding residence lifetimes are therefore 20 and 14 s. Temperature rise results in the increase in rate constants and concomitant Table 3. Temperature dependence of parameters describing volume integrals of autocorrelation peaks for 15NH4þ ions within d[G4(T4G4)3] T (K) f 283 293 303 313 283 293 303 313 283 293 303 313 Binding site I 0.31 ( 0.01) 0.27 ( 0.02) 0.26 ( 0.02) 0.3–0.6 Binding site O1 0.37 ( 0.02) 0.37 ( 0.02) 0.31 ( 0.02) 0.4–1.0 Binding site O2 0.32 ( 0.02) 0.33 ( 0.02) 0.38 ( 0.03) 0.4–0.8 d1 (s 1) 9.1 ( 0.7) 10.2 ( 1.4) 8.0 ( 0.9) 4.0–8.0 8.8 ( 0.8) 7.5 ( 0.5) 8.7 ( 0.7) 4.0–7.0 15.8 ( 1.8) 14.1 ( 1.7) 10.9 ( 1.0) 8.0–14.0 d2 (s 1) 0.32 ( 0.04) 0.57 ( 0.06) 0.81 ( 0.06) 0.4–1.3 0.76 ( 0.07) 0.81 ( 0.06) 1.32 ( 0.06) 0.0–2.0 1.30 ( 0.07) 1.82 ( 0.09) 2.48 ( 0.11) 1.5–3.5 RMSD 0.00178 0.00356 0.00166 40.00328 0.00279 0.00170 0.00184 40.00220 0.00384 0.00384 0.00188 40.00156 Optimized values for fraction f and rate constants d1 and d2 were obtained through iterative ﬁtting of experimental data to Equation (3). Quality of the individual ﬁts is expressed as RMSD. Errors of individual f, d1 and d2 values are given in brackets. Figure 7. Arrhenius plot for O1I and O2I movements. Red open circles and black ﬁlled squares represent the experimental points for the cross- peaks O1I and O2I, respectively.