Tim W. T. Tsai

Steric zipper formed by hydrophobic peptide fragment of Syrian hamster prion protein.

Steric zippers, where the residues of two neighboring β-sheet layers are tightly interdigitated, have been proposed as fundamental structural units of amyloid fibrils by Eisenberg and co-workers. The steric zipper formed by polypeptides containing the palindromic sequence AGAAAAGA has a distinctive feature that the distance between two interdigitated β-sheet layers is comparable to the interstrand distance of the individual β-sheet. This structural motif is of great interest in the study of prion disease because the AGAAAAGA sequence is highly conserved in prion proteins of different species. In this work, the amyloid fibrils formed by the polypeptides of PrP(113-127), viz. Ac-AGAAAAGAVVGGLGG-NH(2), are taken as the model compound to investigate the biophysical principles governing the steric zipper formation. The target fibrils adopt the structural motif of class 7 steric zipper, which is formed by stacking of antiparallel β-sheet layers with residue 117 + k forming backbone hydrogen bonds to residue 120 - k. Implication of our results in the infectivity of scrapie prion is briefly discussed.

Calcium-43 NMR studies of polymorphic transition of calcite to aragonite.

Phase transformation between calcite and aragonite is an important issue in biomineralization. To shed more light on the mechanism of this process at the molecular level, we employ solid-state (43)Ca NMR to study the phase transformation from calcite to aragonite as regulated by magnesium ions, with (43)Ca enrichment at a level of 6%. Using the gas diffusion approach, the phase of Mg-calcite is formed initially and the system subsequently transforms to aragonite as the reaction time proceeds. Our (43)Ca solid-state NMR data support the dissolution-recrystallization mechanism for the calcite to aragonite transition. We find that the (43)Ca NMR parameters of Mg-calcite are very similar to those of pure calcite. Under the high-resolution condition provided by magic-angle spinning at 4 kHz, we can monitor the variation of the (43)Ca NMR parameters of the aragonite signals for the samples obtained at different reaction times. Our data suggest that in the presence of a significant amount of Mg(2+) ions, aragonite is the most stable polymorph of calcium carbonate. The initial precipitated crystallites of aragonite have spine-like morphology, for which the (43)Ca spin-lattice relaxation data indicate that the ions in the lattice have considerable motional dynamics. As the crystallinity of aragonite improves further, the (43)Ca T(1) parameter of the aragonite phase changes considerably and becomes very similar to that obtained for pure aragonite. For the first time, the difference in crystal morphologies and crystallinity of the aragonite phase has been traced down to the subtle difference in the motional dynamics at the molecular level.

Solid-state P-31 NMR study of the formation of hydroxyapatite in the presence of glutaric acid.

We demonstrate that glutaric acid can be used to prepare nanorods of hydroxyapatite under hydrothermal condition at 100 °C with a Ca2+:glutaric acid molar ratio of 1:4. Frequency‐switched Lee–Goldburg irradiation is employed to obtain high‐resolution 31P{1H} correlation spectra of the reaction mixture at two different reaction periods, from which it is shown that octacalcium phosphate is the precursor phase of the final hydroxyapatite product. In addition, the spectra show that a substantial amount of water molecules is trapped between the glutaric acid and the hydroxyapatite surface, indicating that water molecules may play a prominent role in the noncovalent interaction of the glutaric acid and the HAp surface. Copyright

Chapter 1 - Recent Progress in the Solid-State NMR Studies of Biomineralization

Abstract Biomineralization is a challenging research area because it is extremely difficult to study the interaction between two dissimilar organic and inorganic nanophases. In this review, we have briefly discussed some advanced solid-state NMR techniques developed for the study of biominerals or related in vitro model systems. The P-31 NMR parameters of a list of model compounds have been compiled. These data should be useful for analyzing the NMR spectra of biominerals. We intend to provide an account of how one can employ the state-of-the-art solid-state NMR techniques to extract valuable structural information of biominerals that would be difficult to obtain otherwise.

Hydrogen Bond Formation between Citrate and Phosphate Ions in Spherulites of Fluorapatite

Samples of carboxylate-fluorapatite are prepared with citric, tricarballylic, and glutaric acids under hydrothermal conditions. The size of the hexagonal rods differs significantly for the three samples, of which the citric-acid sample exhibits the smallest dimension along the [h00] direction. The solid-state NMR data reveal that all the citrate molecules of citrate-fluorapatite are in direct contact with the fluorapatite surface and that there are at least two binding modes accounting for the interaction between citrate and fluorapatite surface. In addition to the electrostatic interaction between the carboxylate carbons and the calcium ions, some citrate molecules also form hydrogen bond between the hydroxyl group of citrate and the orthophosphate ion of fluorapatite. This hydrogen-bond interaction is highly ordered and may play an important role in the formation of the spherulites.

Time displacement rotational echo double resonance: heteronuclear dipolar recoupling with suppression of homonuclear interaction under fast magic-angle spinning.

We have developed a novel variant of REDOR which is applicable to multiple-spin systems without proton decoupling. The pulse sequence is constructed based on a systematic time displacement of the pi pulses of the conventional REDOR sequence. This so-called time displacement REDOR (td-REDOR) is insensitive to the effect of homonuclear dipole-dipole interaction when the higher order effects are negligible. The validity of td-REDOR has been verified experimentally by the P-31{C-13} measurements on glyphosate at a spinning frequency of 25 kHz. The experimental dephasing curve is in favorable agreement with the simulation data without considering the homonuclear dipole-dipole interactions.

Compensated DRAMA sequence for homonuclear dipolar recoupling under magic-angle spinning.

The DRAMA sequence has been considered as the milestone in the development of homonuclear dipolar recoupling. Although it has a high efficiency for double-quantum excitation in spin 1/2 systems, it is seldom used today for real applications because of its susceptibility to the deteriorating effects of chemical shift anisotropy and resonance offsets. We show in this work that the practicability of DRAMA can be greatly enhanced by incorporating four pi pulses with XY-4 phases into the basic DRAMA cycles. Average Hamiltonian theory is used to evaluate the performance of the resulting pulse sequence with respect to the compensation of chemical shift anisotropy. Numerical simulations and experimental measurements on hydroxyapatite indeed show that the performance of DRAMA-XY4 is very satisfying for 31P DQ excitation, provided that the resonance offset is within the range of [-4, 4]kHz.

Rotational echo double resonance without proton decoupling under fast spinning condition

We show that rotational echo double resonance (REDOR) experiments can be carried out without proton decoupling under the conditions of fast spinning and strong rf field. Numerical simulations on a five-spin systems show that no significant attenuation of the reference signal (S(0)) is observed at a spin rate of 25 kHz, provided that the rf power is larger than 100 kHz. This approach has been validated by (31)P{(13)C} REDOR measurements on isotopically labeled glyphosate. The obtained van Vlecks second moment is in favorable agreement with the value calculated based on the crystal structure.

Measurements of 13C Multiple-Quantum Coherences in Amyloid Fibrils under Magic-Angle Spinning

The excitation and detection of high-order multiple quantum coherences among (13)C nuclear spins are demonstrated in the samples of [1-(13)C]-L-alanine and (13)C labeled amyloid fibrils at a spinning frequency of 20 kHz. The technique is based on the double-quantum average Hamiltonian prepared by the DRAMA-XY4 pulse sequence. Empirically, we find that multiple supercycles are required to suppress the higher-order effects for real applications. Measurements for the fibril samples formed by the polypeptides of PrP(113-127) provide the first solid-state NMR evidence for the stacking of multiple β-sheet layers at the structural core of amyloid fibrils.