Development and application of multidimensional dipolar recoupling methods for distance measurement in nucleic acids.

摘要

Distance information is encoded in solid state NMR spectra in the form of dipolar couplings between nuclear magnetic moments. This distance information can be resolved from other, stronger interactions by combining magic angle spinning with radio frequency pulse sequences. Dipolar recoupling with a windowless sequence (DRAWS) has been developed as a distance measurement technique and is here demonstrated as a method for distance measurements in nucleic acids. The experiment is relatively insensitive to chemical shift parameters, pulse imperfections, and radio frequency inhomogeneity. It is applied to a series of doubly ;Limitations of this technique are addressed by developing a two-dimensional application which can be applied to multiply labeled samples. Sideband suppression eliminates the dispersive peak components usually observed in multi-dimensional magic angle spinning spectra. This allows the use of time reversal techniques for hypercomplex data acquisition. Dipolar couplings between nuclei lead to crosspeaks in the two-dimensional spectrum. The time development of the crosspeaks in a series of two-dimensional experiments with progressively longer mixing times is shown to be related to the dipolar couplings and hence to the internuclear distances. The crosspeaks also encode information about the magnetization transfer pathway as they are negative for direct coherence transfer and additional steps lead to alternation of the sign. A theoretical description of this behavior is developed.;The limitations of this two-dimensional experiment for internuclear distance measurements are explored in a series of experiments on five isotopically labeled, crystalline cytidine samples. Different labeling schemes are used to provide samples with the same nominal structure, but very different spin system geometries and sizes. The experimental results are analyzed by direct simulation to determine the internuclear distances and the effectiveness of the experiment at resolving structural problems commonly found in studies of nucleic acids. The effects of indirect magnetization transfer on the resultant distance measurements are discussed in detail.