Preliminary In Research Of Experimental Methods For Proteins By Solid-state MAS NMR

Solid-state magic angle spinning nuclear magnetic resonance (MAS NMR) has been rapidly developed in the last decade and widely used in the studies of proteins. It can resolve the atomic three-dimensional structures and dynamics of proteins and interactions between proteins and other molecules; it does not require protein crystallization or dissolution. Thus, solid-state NMR is regarded as a very promising technique for studying insoluble proteins such as membrane proteins.Although having made some progresses in membrane proteins and amyloid protein fibrils, solid-state NMR is still less efficient compared to X-ray crystallography and liquid-state NMR. The obstacle which limits its application in proteins is the degeneracy of experimental sensitivity and spectral resolution. While the sensitivity is limited by the detection of low-γ13C or15N, the detected NMR signals will be further attenuated by the multiple steps of magnetization transfer in multi-dimensional experiments. Besides the anisotropic interactions in solids, the resolution is mainly influenced by the heterogeneity of protein samples. Consequently, new methods to enhance the magnetization transfer efficiency and analytical strategies to optimize the preparation of protein samples are very important for the studies of proteins by solid-state NMR.Based on these concerns, this dissertation mainly focused on the development of magnetization transfer methods, e.g. hetero-nuclear and homo-nuclear dipolar recoupling methods, which would be beneficial to multi-dimensional correlation experiments. In the meantime, it analyzed protein samples by solid-state NMR in order to promote the screening of conditions for sample preparation and thus gained protein samples with high resolution which were appropriate for solid-state NMR.The hetero-nuclear dipolar recoupling between15N and13C was very important for multi-dimensional SSNMR experiments. Firstly, via sinusoidal amplitude modulation of13C radio-frequency (RF) fields, we achieved selectively dual-band15N-13C hetero-nuclear magnetization transfer and thus simultaneously established intra-residual correlation and inter-residual correlation of protein backbones in a single experiment. Compared to the regular methods for dual-band selectivity, our method was more beneficial to multi-dimensional experiments due to its better selectivity, stability and less demand on1H decoupling RF fields.Secondly, we investigated the influence of simultaneous phase inversion on15N-13C cross polarization (CP). It was found that asymmetrically simultaneous phase inversion could evidently improve the stability of15N-13C hetero-nuclear magnetization transfer, and might also increase the transfer efficiency. The asymmetrically simultaneous phase-inversion CP would contribute to those low-sensitivity or long-time multi-dimensional experiments.We also probed the probability of RN symmetry-based sequences used for band-selective13C-13C homo-nuclear dipolar recoupling without1H decoupling. With the help of computer simulations, we obtained several eligible RN conditions and then verified their effectiveness of band-selectivity without1H decoupling in standard compounds. These RN methods would improve the selectivity of magnetization transfer between13Cα-13Cβ and reduce the risk of protein deterioration induced by sample heating under high1H decoupling power.In order to promote the preparation of high-resolution protein samples, we analyzed the heterogeneity of samples via NMR spectra, and summed up to an analytical strategy-from1D15N spectra of a few15N labeled protein samples to2D13C-13C/15N-13Cα correlation spectra of a few U-13C,15N labeled protein samples by solid-state NMR, which was suitable for optimization of protein samples.