Constraints on the structure of (CUG)97 RNA from magic-angle-spinning solid-state NMR spectroscopy.
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3' Untranslated Regions↗
Biomedical subjects
Kerstin Riedel
Publications and source records attributed to Kerstin Riedel.
Tailoring broadband inversion pulses for MAS solid state NMR.
A simple approach is demonstrated for designing optimised broadband inversion pulses for MAS solid state NMR studies of biological systems. The method involves a two step numerical optimisation procedure and takes into account experimental requirements such as the pulse length, resonance offset range and extent of H(1) inhomogeneity compensation needed. A simulated annealing protocol is used initially to find appropriate values for the parameters that define the well known tanh/tan adiabatic pulse such that a satisfactory spin inversion is achieved with minimum RF field strength. This information is then used in the subsequent stage of refinement where the RF pulse characteristics are further tailored via a local optimisation procedure without imposing any restrictions on the amplitude and frequency modulation profiles. We demonstrate that this approach constitutes a generally applicable tool for obtaining pulses with good inversion characteristics. At moderate MAS frequencies the efficacy of the method is experimentally demonstrated for generating double-quantum NMR spectra via the zero-quantum dipolar recoupling scheme RFDR.
Carbon Isotopes↗
Solid state NMR at high magic angle spinning frequencies: dipolar chemical shift correlation with adiabatic inversion pulse based RF pulse schemes.
The efficacy of hetero- and homonuclear dipolar recoupling employing tanh/tan adiabatic inversion pulse based RF pulse schemes has been examined at high magic angle spinning (MAS) frequencies via numerical simulations and experimental measurements. An approach for minimising the recoupling RF power level is presented, taking into consideration the spinning speed, the range of resonance offsets and H(1) inhomogeneities and the available RF field strength. This involves the tailoring of the frequency and amplitude modulation profiles of the inversion pulses. The applicability of tanh/tan pulse based dipolar recoupling schemes to spinning speed regimes where the performance with conventional rectangular pulses may not be satisfactory is demonstrated.
Carbon Isotopes↗
Impact of reduced O-acetylserine(thiol)lyase isoform contents on potato plant metabolism.
Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to down-regulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H(2)S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed.
Carbon-Oxygen Lyases↗
TEDOR with adiabatic inversion pulses: Resonance assignments of 13C/15N labelled RNAs.
We have examined via numerical simulations the performance characteristics of different 15N RF pulse schemes employed in the transferred echo double resonance (TEDOR) experimental protocol for generating 13C-15N dipolar chemical shift correlation spectra of isotopically labelled biological systems at moderate MAS frequencies (omega(r) approximately 10 kHz). With an 15N field strength of approximately 30-35 kHz that is typically available in 5 mm triple resonance MAS NMR probes, it is shown that a robust TEDOR sequence with significant tolerance to experimental imperfections sa as H1 inhomogeneity and resonance offsets can be effectively implemented using adiabatic heteronuclear dipolar recoupling pulse schemes. TEDOR-based 15N-13C and 15N-13C-13C chemical shift correlation experiments were carried out for obtaining 13C and 15N resonance assignments of an RNA composed of 97 (CUG) repeats which has been implicated in the neuromuscular disease myotonic dystrophy.
Carbon Isotopes↗
Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy.
It is demonstrated that the spatial proximity of (1)H nuclei in hydrogen bonded base-pairs in RNAs can be conveniently mapped via magic angle spinning solid state NMR experiments involving proton spin diffusion driven chemical shift correlation of low gamma nuclei such as the imino and amino nitrogens of nucleic acid bases. As different canonical and non-canonical base-pairing schemes encountered in nucleic acids are characterised by topologically different networks of proton dipolar couplings, different base-pairing schemes lead to characteristic cross-peak intensity patterns in such correlation spectra. The method was employed in a study of a 100 kDa RNA composed of 97 CUG repeats, or (CUG)(97) that has been implicated in the neuromuscular disease myotonic dystrophy. (15)N-(15)N chemical shift correlation studies confirm the presence of Watson-Crick GC base pairs in (CUG)(97).
Base Pairing↗
Homonuclear chemical shift correlation in rotating solids via RNnu n symmetry-based adiabatic RF pulse schemes.
The efficacy of RN(nu) (n) symmetry-based adiabatic Zero-Quantum (ZQ) dipolar recoupling schemes for obtaining chemical shift correlation data at moderate magic angle spinning frequencies has been evaluated. RN(nu) (n) sequences generally employ basic inversion elements that correspond to a net 180 degrees rotation about the rotating frame x-axis. It is shown here via numerical simulations and experimental measurements that it is also possible to achieve efficient ZQ dipolar recoupling via RN(nu) (n) schemes employing adiabatic pulses. Such an approach was successfully used for obtaining (13)C chemical shift correlation spectra of a uniformly labelled sample of (CUG)(97) - a triplet repeat expansion RNA that has been implicated in the neuromuscular disease myotonic dystrophy. An analysis of the (13)C sugar carbon chemical shifts suggests, in agreement with our recent (15)N MAS-NMR studies, that this RNA adopts an Alpha-helical conformation.
Nuclear Magnetic Resonance, Biomolecular↗