Biomolecular ligands screening using radiation damping difference WaterLOGSY spectroscopy.

Water-ligand observed via gradient spectroscopy (WaterLOGSY) is a widely used nuclear magnetic resonance method for ligand screening. The crucial procedure for the effectiveness of WaterLOGSY is selective excitation of the water resonance. The selective excitation is conventionally achieved by using long selective pulse, which causes partial saturation of the water magnetization leading to reduction of sensitivity, in addition to time consuming and error prone. Therefore, many improvements have been made to enhance the sensitivity and robustness of the method. Here we propose an alternative selective excitation scheme for WaterLOGSY by utilizing radiation damping effect. The pulse scheme starts simply with a hard inversion pulse, instead of selective pulse or pulse train, followed by a pulse field gradient to control the radiation damping effect. The rest parts of the pulse scheme are similar to conventional WaterLOGSY. When the gradient pulse is applied immediately after the inversion pulse, the radiation damping effect is suppressed, and all of the magnetization is inversed. When the gradient pulse and the inversion pulse are about 10-20 ms apart, the radiation damping effect remains active and drives the water magnetization toward +z-axis, resulting in selective non-inversion of the water magnetization. By taking the differences of the spectra obtained under these two conditions, one should get the result of WaterLOGSY. The method is demonstrated to be simple, robust and sensitive for ligand screening.

Conformational toggling of yeast iso-1-cytochrome C in the oxidized and reduced states.

To convert cyt c into a peroxidase-like metalloenzyme, the P71H mutant was designed to introduce a distal histidine. Unexpectedly, its peroxidase activity was found even lower than that of the native, and that the axial ligation of heme iron was changed to His71/His18 in the oxidized state, while to Met80/His18 in the reduced state, characterized by UV-visible, circular dichroism, and resonance Raman spectroscopy. To further probe the functional importance of Pro71 in oxidation state dependent conformational changes occurred in cyt c, the solution structures of P71H mutant in both oxidation states were determined. The structures indicate that the half molecule of cyt c (aa 50-102) presents a kind of "zigzag riveting ruler" structure, residues at certain positions of this region such as Pro71, Lys73 can move a big distance by altering the tertiary structure while maintaining the secondary structures. This finding provides a molecular insight into conformational toggling in different oxidation states of cyt c that is principle significance to its biological functions in electron transfer and apoptosis. Structural analysis also reveals that Pro71 functions as a key hydrophobic patch in the folding of the polypeptide of the region (aa 50-102), to prevent heme pocket from the solvent.

The impact of pulse duration on composite WATERGATE pulse.

As an effective method for solvent suppression, WATERGATE is widely used in high resolution NMR spectroscopy. It is usually composed of a number of pulses separated by constant intervals. However, theoretical and experimental analyses indicate that narrower bandwidth and lower intensities around the secondary suppression points occur in the excitation profile of the composite WATERGATE. The excitation profile distortion is caused by the chemical shift evolution during the RF pulses. The higher the ratio of pulse duration to the inter-pulse delay is, the severer the profile distorts. Therefore, in high magnetic fields, the effect will be serious when WATERGATE is applied to some biological samples whose resonances distribute over a wide range. As can be seen obviously by applying WATERGATE to detect a RNA-protein mixture sample in an 800 MHz spectrometer, the resonances of the imino protons were partially suppressed by showing decreased intensities, though the intended secondary suppression points were set far away from them. In this article, we proposed an optimized WATERGATE that could effectively compensate the chemical shift evolution during the RF pulses, and relieve the excitation profile distortion. The optimized experiment will be a good way to retain the imino signal intensities when WATERGATE is applied to detect the RNA samples in high magnetic field.

Gridding and fast Fourier transformation on non-uniformly sparse sampled multidimensional NMR data.

For multidimensional NMR method, indirect dimensional non-uniform sparse sampling can dramatically shorten acquisition time of the experiments. However, the non-uniformly sampled NMR data cannot be processed directly using fast Fourier transform (FFT). We show that the non-uniformly sampled NMR data can be reconstructed to Cartesian grid with the gridding method that has been wide applied in MRI, and sequentially be processed using FFT. The proposed gridding-FFT (GFFT) method increases the processing speed sharply compared with the previously proposed non-uniform Fourier Transform, and may speed up application of the non-uniform sparse sampling approaches.