Incubation of Amyloidogenic Peptides in Reverse Micelles Allow Active Control of Oligomer Size and Study of Protein-Protein Interactions.

Studies of the structure and dynamics of oligomeric aggregates of amyloidogenic peptides pose challenges due to their transient nature. This concept article provides a brief overview of various nucleation mechanisms with reference to the classical nucleation theory and illustrates the advantages of incubating amyloidogenic peptides in reverse micelles (RMs). The use of RMs not only facilitates size regulation of oligomeric aggregates but also provides an avenue to explore protein-protein interactions among the oligomeric aggregates of various amyloidogenic peptides. Additionally, we envision the feasibility of preparing brain tissue-derived oligomeric aggregates using RMs, potentially advancing the development of monoclonal antibodies with enhanced potency against these pathological species in vivo.

Solid state NMR - An indispensable tool in organic-inorganic biocomposite characterization; refining the structure of octacalcium phosphate composites with the linear metabolic di-acids succinate and adipate.

Octacalcium phosphate (OCP; Ca8(HPO4)2(PO4)4. 5H2O) is a plausible precursor phase of biological hydroxyapatite, which composites with a number of biologically relevant organic metabolites. Widely used material science physicochemical structure determination techniques successfully characterize the mineral component of these composites but leave details of the structure, and interactions with mineral, of the organic component almost completely obscure. The metabolic linear di-acids succinate (SUC) and adipate (ADI) differentially expand the hydrated (100) layer of OCP. 13C13C correlation (proton driven spin diffusion, PDSD) experiments on OCP composited with (U-13C4)-SUC, and (U13C6)-ADI, show that the two di-acids per unit cell adopt non-centrosymmetric but mutually identical structures. 13C{31P}, rotational echo double resonance (REDOR) shows that one end of each linear di-acid is displaced further from the surface of the apatitic OCP layer relative to the other end. Overall the results indicate two di-acids per unit cell disposed perpendicularly across the OCP hydrated layer with one carboxylate of each di-acid substituting a hydrated surface OCP phosphate group. This study re-affirms the unique advantages of ssNMR in elucidating structural details of organic-inorganic biocomposites, and thereby mechanisms underlying the roles of small metabolites in influencing biomineralization mechanisms and outcomes.

Zinc ion rapidly induces toxic, off-pathway amyloid-ß oligomers distinct from amyloid-ß derived diffusible ligands in Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the elderly. Zinc (Zn) ion interacts with the pathogenic hallmark, amyloid-ß (Aß), and is enriched in senile plaques in brain of AD patients. To understand Zn-chelated Aß (ZnAß) species, here we systematically characterized ZnAß aggregates by incubating equimolar Aß with Zn. We found ZnAß40 and ZnAß42 both form spherical oligomers with a diameter of ~12-14 nm composed of reduced ß-sheet content. Oligomer assembly examined by analytical ultracentrifugation, hydrophobic exposure by BisANS spectra, and immunoreactivity of ZnAß and Aß derived diffusible ligands (ADDLs) are distinct. The site-specific 13C labeled solid-state NMR spectra showed that ZnAß40 adopts ß-sheet structure as in Aß40 fibrils. Interestingly, removal of Zn by EDTA rapidly shifted the equilibrium back to fibrillization pathway with a faster kinetics. Moreover, ZnAß oligomers have stronger toxicity than ADDLs by cell viability and cytotoxicity assays. The ex vivo study showed that ZnAß oligomers potently inhibited hippocampal LTP in the wild-type C57BL/6JNarl mice. Finally, we demonstrated that ZnAß oligomers stimulate hippocampal microglia activation in an acute Aß-injected model. Overall, our study demonstrates that ZnAß rapidly form toxic and distinct off-pathway oligomers. The finding provides a potential target for AD therapeutic development.

Structural characterization of fluoride species in shark teeth.

In shark teeth we have identified the species fluorapatite, hydroxyfluorapatite and its defect site, calcium fluoride, and potassium fluoride. Their relative amounts in teeth at different development stages have been quantified. Calcium fluoride and potassium fluoride may be associated with the fluoridation mechanism in shark teeth.

Is Mg-stabilized amorphous calcium carbonate a homogeneous mixture of amorphous magnesium carbonate and amorphous calcium carbonate?

We find two types of carbonate ions in Mg stabilized amorphous calcium carbonate (Mg-ACC), whose short-range orders are identical to those of ACC and amorphous magnesium carbonate (AMC). Mg-ACC comprises a homogeneous mixture of the nano-clusters of ACC and AMC. Their relative amount varies systematically at different pH.

Aggregation of Beta-Amyloid Peptides Proximal to Zwitterionic Lipid Bilayers.

One of the hallmarks of Alzheimers disease is the deposition of amyloid plaques, which consist of ß-amyloid (Aß) peptides in fibrillar states. Nonfibrillar Aß aggregates have been considered as an important intermediate in the pathway of fibrillization, but little is known about the formation mechanism. The on-pathway ß-sheet intermediates of Aß40 peptides can be trapped by incubating the peptides in liposomes formed by zwitterionic lipids. The aggregates of Aß40 peptides have been prepared at a peptide concentration of less than 10 µm. Solid-state NMR spectroscopy data show that the backbone conformation of the aggregates is almost identical to that of the fibrils formed in free solution. In contrast to anionic lipids, zwitterionic lipids, which are typical of neuronal soma, did not induce any significant conformational difference in Aß40 fibrils. This liposome-Aß system may serve as a useful model to study the fibril formation mechanism.

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.

Constant time tensor correlation experiments by non-gamma-encoded recoupling pulse sequences.

Constant-time tensor correlation under magic-angle spinning conditions is an important technique in solid-state nuclear magnetic resonance spectroscopy for the measurements of backbone or side-chain torsion angles of polypeptides and proteins. We introduce a general method for the design of constant-time tensor correlation experiments under magic-angle spinning. Our method requires that the amplitude of the average Hamiltonian must depend on all the three Euler angles bringing the principal axis system to the rotor-fixed frame, which is commonly referred to as non-gamma encoding. We abbreviate this novel approach as COrrelation of Non-Gamma-Encoded Experiment (CONGEE), which exploits the orientation-dependence of non-gamma-encoded sequences with respect to the magic-angle rotation axis. By manipulating the relative orientation of the average Hamiltonians created by two non-gamma-encoded sequences, one can obtain a modulation of the detected signal, from which the structural information can be extracted when the tensor orientations relative to the molecular frame are known. CONGEE has a prominent feature that the number of rf pulses and the total pulse sequence duration can be maintained to be constant so that for torsion angle determination the effects of systematic errors owing to the experimental imperfections and/or T(2) effects could be minimized. As a proof of concept, we illustrate the utility of CONGEE in the correlation between the C' chemical shift tensor and the C(α)-H(α) dipolar tensor for the backbone psi angle determination. In addition to a detailed theoretical analysis, numerical simulations and experiments measured for [U-(13)C, (15)N]-L-alanine and N-acetyl-[U-(13)C, (15)N]-D,L-valine are used to validate our approach at a spinning frequency of 20 kHz.

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.

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 ß-sheet layers at the structural core of amyloid fibrils.

Manipulating the nanostructure of organogels generated from molecules with a 3-dimensional truxene core.

Two new truxene-based 3-dimensional (3-D) molecules self-assembled in cyclohexane to give organogels with vesicular and fibrillar nano-morphologies governed by the substitution pattern of the peripheral alkyl amido side-chains grafted onto the rigid 3-D core.

Solid-state NMR techniques for the structural determination of amyloid fibrils.

This review discusses the solid-state NMR techniques developed for the study of amyloid fibrils. Literature up to the end of 2010 has been surveyed and the materials are organized according to five categories, viz. homonuclear dipolar recoupling and polarization transfer via J-coupling, heteronuclear dipolar recoupling, correlation spectroscopy, recoupling of chemical shift anisotropy, and tensor correlation. Our emphasis is on the NMR techniques and their practical aspects. The biological implications of the results obtained for amyloid fibrils are only briefly discussed. Our main objective is to showcase the power of NMR in the study of biological unoriented solids.

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.

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

Steric zippers, where the residues of two neighboring ß-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 ß-sheet layers is comparable to the interstrand distance of the individual ß-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 ß-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.

Internal symmetry of basic elements in symmetry-based recoupling sequences under magic-angle spinning.

In solid-state NMR, many powerful pulse sequences under the condition of magic-angle spinning can be analyzed on the basis of the C- and R-sequences developed by Levitt and co-workers. It has been speculated for some years that the basic elements commonly used in symmetry-based recoupling pulse sequences have certain kind of internal symmetries. We show by a detailed analysis that a set of internal selection rules does exist for many basic elements. These internal selection rules may allow a more versatile design of CN(n)(ν) or RN(n)(ν) sequences when n is an integer or half-integer multiple of N. As an illustration, we have derived the symmetry arguments to rationalize the observation that the C-REDOR pulse sequence can suppress homonuclear dipole-dipole interaction, leading to the design of new windowed basic elements usable for heteronuclear dipolar recoupling with active suppression of homonuclear dipole-dipole interaction. Numerical simulations and experiments measured for [U-(13)C,(15)N]-L-alanine have been used to validate our approach. On a more general note, the symmetry rules discussed in this work can also be applied for the design of supercycles.

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 Vleck's second moment is in favorable agreement with the value calculated based on the crystal structure.

Solid-state P-31 NMR study of octacalcium phosphate incorporated with succinate.

Octacalcium phosphate (OCP) is an important model compound in the study of biomineralization. The octacalcium phosphate-succinate (OCPS) compound is prepared and characterized by (31)P solid-state NMR spectroscopy. Taking advantage of the fact that the crystal structures of OCP and OCPS are very similar, an NMR strategy based on the (31)P homonuclear double-quantum spectroscopy is developed to assign all the peaks observed in the (31)P magic-angle spinning spectrum of OCPS. On the basis of our experimental data, the molecular formula of OCPS is determined to be Ca(7.81)(HPO(4))(1.82)(PO(4))(3.61)(succinate)(0.56).zH(2)O, where z

Molecular structure of amyloid fibrils formed by residues 127 to 147 of the human prion protein.

Amyloid fibrils are filamentous and insoluble forms of peptides or proteins. Proline has long been considered to be incompatible with the cross-beta structural motif of amyloid fibrils. On the basis of solid-state NMR spectroscopy data, we present a structural model of an in-register parallel beta sheet for the amyloid fibrils formed from a human prion protein fragment, huPrP(127-47). We have developed a simple solid-state NMR spectroscopy technique to identify solvent-protected backbone amide protons in a H/D exchange experiment without disaggregating the amyloid fibrils, from which we find that proline residue P(137) does not disrupt the beta-sheet structure from G(127) to G(142). We suggest that the resultant kink at P(137) generates a twist between adjacent peptide strands to maintain hydrogen bonding in the beta-sheet regions flanking the P(137) residue. Although proline can be well integrated into the cross-beta structure of amyloid fibrils, the kink formed at the position of the proline residue will considerably weaken the hydrogen bonding between the neighboring strands, especially when the mutation site is near the central region of a beta sheet.

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.

Heteronuclear dipolar recoupling of half-integer quadrupole nuclei under fast magic angle spinning.

An experimental method for the heteronuclear dipolar recoupling of half-integer quadrupole nuclei is proposed. The idea is to manipulate the central transition based on the recoupling technique of spin-polarization-inversion rotary resonance. This method allows the extraction of structural parameters under fast magic-angle spinning. Its validity has been examined by the average Hamiltonian theory and numerical simulations. The initial rotational-echo dephasing arising from the dipolar evolution can be approximated by a parabolic function, from which the heteronuclear van Vleck second moment can be estimated. A factor, estimated from two-spin simulations, is required to account for the effects of the quadrupolar coupling and is rather independent of the geometry and the orders of the spin systems. Our method can facilitate the structural characterization of materials containing half-integer quadrupole nuclei under high-resolution condition. Experimental verification has been carried out on two aluminophosphate systems, namely, AlPO(4)-5 and AlPO(4)-11.