Popis: |
Figure 1. Relative peak intensities in the inversion recovery spectra for the imino proton (open circle) and base proton (gray circle) resonances as a function of delay time. Solid and dash lines indicate the best fit to single and double exponential functions, respectively. The Tetrahymena group ribozyme is the valuable model for investigating the principle of RNA folding, structure, and function. The Tetrahymena ribozyme catalyze a phosphoryl transfer reaction with a rate enhancement of 10-fold over the uncatalyzed reaction. Binding of the Tetrahymena ribozyme’s oligonucleotide substrate involves P1 duplex formation with the ribozyme’s internal guide sequence (IGS) to give an open complex, followed by docking of the P1 duplex into the catalytic core via tertiary interactions to give a closed complex. The monitoring on the structural or biophysical change between the docked and undocked states of the ribozyme is one of the good methods to investigate its dynamics and/or folding. The exchange of the imino protons with solvent water, which can be measured by NMR, implicates not only the base-pair opening rate but also the solvent accessibility. The solvent accessibility provides the important information about the structure feature of nucleic acids. Recently, the Hydrogen/Deuterium exchange study of ribozyme reported that some imino proton resonances near 12.8 and 13.4 ppm had the exchange times of 3.89 and 5.18 hours, respectively, indicating that these imino protons slowly exchanged to solvents. The water magnetization transfer experiment is a useful NMR method for the exchange time measurement of the imino protons in nucleic acids. This approach required the measure the apparent T1 (T1a) time of the imino proton signals, which could be determined by the inversion-recovery experiment. The T1a time is expressed by equation, 1/T1a = R1a = 1/T1 + kex, where T1 is the dipolar relaxation time and kex is the exchange rate constant. We performed the inversion-recovery experiment to measure the T1a time of the imino proton signals of the ribozyme at 35 C. Surprisingly, these inversion-recovery data were not fitted by a single exponential equation but fitted well by a double exponential function (Fig. 1). This unusual double exponential relaxation has been reported in the NMR study of the short RNA duplex, which might result from partial aggregation of RNA duplex. The imino proton resonances of the ribozyme are shown like the several broad peaks, but these resonances are the mixture of at least one hundred of imino resonances. Thus, this double exponential function of the inversion-recovery data is thought to be the summation of the T1a values of lots of imino protons. However, this hypothesis cannot explain the similar patterns of the non-exchangeable resonances (Fig. 1) because every base proton, except A-H2, has the similar T1 (= T1a) value. In order to explain this result, the Solomon equation, which is the best theory to explain the NOE effect by selective inversion, is introduced. First, we consider two-spin model, where spin 1 is selectively inverted at t = 0. This relaxation can be represented by Solomon equations. |