HETEROTOCSY-BASED EXPERIMENTS FOR MEASURING HETERONUCLEAR RELAXATION IN NUCLEIC-ACIDS AND PROTEINS

While both P-31 and Cd-113 are present at locations of interest in many different macromolecular systems, heteronuclear-detected relaxation measurements on these nuclei have been restrained by limitations in either resolution or signal-to-noise ratio. We have developed heteroTOCSY-based methods to overcome both of these problems. Two-dimensional versions of these experiments were utilized to measure P-31 T-1 and T-2 values in DNA oligonucleotides; the additional resolution offered by a second dimension allowed determination of these values for most of the P-31 resonances in a DNA dodecamer. The results from the experiments indicated that there was little significant variation in T-1 values for the different phosphates in the DNA dodecamer; however, the T-2 values showed a clear pattern, with lower values in the interior of the sequence than at the ends of the helix. Furthermore, a significant correlation between P-31 chemical shifts and T-2 values was observed. One-dimensional, frequency-selective versions of these experiments were also developed for use on systems containing a smaller number of heteronuclear spins. These methods were applied to investigate the heteronuclear relaxation properties of Cd-113 in (113)Cd(2)LAC9(61), a Cys(6)Zn(2) DNA-binding domain. Data from the experiments confirm biochemical evidence that more significant differences occur in metal-protein interactions between the two metal-binding sites than has been previously identified for proteins containing this motif.