Bidirectional band-selective magnetization transfer along the protein backbone doubles the information content of solid-state NMR correlation experiments.

Resonance assignment is the first stage towards solving the structure of a protein. This is normally achieved by the employment of separate inter and intra residue experiments. By utilising the mixed rotation and rotary recoupling (MIRROR) condition it is possible to double the information content through the efficient bidirectional transfer of magnetization from the CO to its adjacent Cα and the Cα of the subsequent amino acid. We have incorporated this into a 3D experiment, a 3D-MIRROR-NCOCA, where correlations present in the 3D spectrum permit the sequential assignment of the protein backbone from a single experiment as we have demonstrated on a microcrystalline preparation of GB3. Furthermore, the low-power requirements of the MIRROR recoupling sequence facilitate the development of a low-power 3D-NCOCA experiment. This has enabled us to realise significant reductions in acquisition times, allowing the acquisition of a single 3D-NCOCA spectrum suitable for a full backbone resonance assignment of GB3 in less than 24 h.

Measurement of 14N quadrupole couplings in biomolecular solids using indirect-detection 14N solid-state NMR with DNP.

The quadrupolar interaction experienced by the spin-1 14N nucleus is known to be extremely sensitive to local structure and dynamics. Furthermore, the 14N isotope is 99.6% naturally abundant, making it an attractive target for characterisation of nitrogen-rich biological molecules by solid-state NMR. In this study, dynamic nuclear polarization (DNP) is used in conjunction with indirect 14N detected solid-state NMR experiments to simultaneously characterise the quadrupolar interaction at multiple 14N sites in the backbone of the microcrystalline protein, GB3. Considerable variation in the quadrupolar interaction (>700 kHz) is observed throughout the protein backbone. The distribution in quadrupolar interactions observed reports on the variation in local backbone conformation and subtle differences in hydrogen-bonding; demonstrating a new route to the structural and dynamic analysis of biomolecules.

C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages.

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.

Effects of diethyldithiocarbamate on the catabolism of tyrosine in the rat brain.

500 mg/kg sodium diethyldithiocarbamate (DDC) and trace quantities of uniformly labelled 14C-tyrosine were administered simultaneously to male albino rats of Porton-Wistar strain of approximately 210 g body weight. Thirty minutes later total radioactivity, the concentration and the specific activity of free tyrosine were increased both in plasma and in the brain by 40% compared with rats untreated with DDC. The incorporation of 14C from 14C-tyrosine into the fraction corresponding to the elution of glutamine-glutamate from the amberlite resin column was 80% less in the brain 30 min after DDC. The exhalation of 14CO2 was depressed by 80% in the first hour after DDC. When 14C-tyrosine was given 3.5 h after DDC the only differences between experimental and control rats were the increased incorporation of 14C into the glutamine-glutamate and aspartate fractions and the increased exhalation of 14CO2 which became significiant in the third and fourth half hour periods after the injection of 14C-tyrosine. From the experiments it is concluded that DDC, an inhibitor of dopamine-beta-hydroxylase, also interferes with the major catabolic pathway of tyrosine.

13C NMR relaxation studies of molecular motion in peptide fragments from human transthyretin.

Natural-abundance 13C T1 and NOE measurements have been made for backbone and side-chain sites in peptide fragments of transthyretin (TTR 10-20, TTR 105-115, and TTR 105-115Met111) at 13C Larmor frequencies of 125 and 75 MHz. These peptides have previously been implicated in the formation of amyloid fibrils. The data were systematically assessed for their consistency with theoretical relaxation parameters derived from models of molecular motion. It was shown that of four models, ranging from simple isotropic motion to one defining internal wobbling of the 13C-1H vector, the "model-free approach" (Lipari and Szabo, J. Am. Chem. Soc. 104, 4546, 1982) was best able to predict the experimental data. These peptides exhibited overall correlation times close to 1 ns. Internal motions with effective correlation times of approximately 0.08 ns were observed for backbone carbon sites, and side-chain carbons exhibited more rapid and less ordered motions. No indication of retarded motion due to the presence of small peptide aggregates was detected, in agreement with reports of the rapid incorporation of these species into amyloid fibrils.

In vivo inhibition of dopamine-beta-hydroxylase in rat adrenals during exposure to carbon disulphide.

Male rats were exposed for a maximum of 4 h to carbon disulphide at atmospheric levels of 1.0-4.0 mg/l and the turnover rates of adrenal dopamine was determined by injecting alpha-methyl-p-tyrosine and measuring the rate at which dopamine disappears. Although the level of exposure was significantly higher than the 30.0 micrograms/l permissible limit, or the average occupational exposure, similar or even higher peak exposure values were reported from the viscose rayon industry. After inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine, adrenal dopamine contents declined at a slower rate in rats exposed to carbon disulphide than in controls. The reduced rate of dopamine metabolism during exposure to carbon disulphide indicates inhibition of dopamine-beta-hydroxylase in vivo. The size of this effect, which could be detected as soon as 30 min after starting the exposure to carbon disulphide, was dose dependent. The rate of dopamine turnover was still reduced 2 h after the end of a single exposure. However at that time, because of the larger dopamine pool present in the adrenals, the amount of dopamine converted per unit of time was again at pre-exposure levels.

Structural analysis of peptide fragment 71-93 of transthyretin by NMR spectroscopy and electron microscopy: insight into amyloid fibril formation.

A peptide corresponding to the amino acid region 71-93 of the plasma protein transthyretin (TTR) has been synthesized to investigate its role in the native folding of the molecule and the possible relationship between mutations in this region and amyloid formation of TTR. In the native structure this fragment includes a beta-strand followed by a short helix and turns back on itself to form part of an antiparallel beta-sheet. Electron microscopy has shown that the peptide is not intrinsically amyloidogenic. NMR spectroscopy has been used to investigate the conformational dependency of the peptide on the solution conditions. Minor populations of peptide showing partial turns were apparent in deuterated dimethyl sulfoxide (DMSO-d6). Some indication of nascent helix between residues 5 and 12 was observed in water, and upon the addition of 20% trifluoroethanol (TFE) the span of helix was confirmed. The intrinsic tendency to form a helical structure between residues 5 and 12 in solution suggests that the helical region, also present in the native crystallographically determined TTR structure at corresponding residues 75-82, is an important folding initiation site. In contrast, the beta-sheet motif observed in the native structure was not detected in solution. It is proposed that mutations in TTR occurring in the helical region result in subtle changes in the TTR structure leading to amyloid fibril formation.

X-ray diffraction studies of fibrils formed from peptide fragments of transthyretin.

Two synthetic peptide fragments of the plasma protein transthyretin (TTR), previously shown to form fibrillar structures in vitro, have been examined using electron microscopy and X-ray diffraction. The fibrils displayed all characteristics of cross beta-sheet conformation with antiparallel strand spacing of 4.7 A and intersheet spacings of 8-10 A as well as reflections indicating further lateral repeating units. A third peptide containing a substitution equivalent to a mutation in TTR known to increase the propensity of TTR to form amyloid was also examined. It also formed fibrils and showed similar cross beta-sheet structure, but with closer intersheet packing than its native equivalent.

1H NMR analysis of fibril-forming peptide fragments of transthyretin.

Peptide fragments of the protein transthyretin, previously shown to form cross beta-sheet amyloid-like fibrils in vitro, were investigated using 1H 1D and 2D NMR techniques. TTR 10-20, TTR 105-115 as well as a substituted analogue, (TTR 105-115Met111) all formed amyloid-like fibrils readily in 20-30% acetonitrile/water at room temperature. It was found that the presence of fibrils in the peptide solutions did not affect the observable NMR spectra, which may have been due to the line-broadening that would be associated with these macromolecular species. 1H NMR spectra were thus representative of the monomeric form of the peptide in solution. Information from D2O exchange, 3JNH-alpha H coupling measurements, temperature coefficients and NOESY experiments suggested that these peptides have some propensity for turn or helix but were predominantly unstructured. There was no indication of the monomeric species existing predominantly in an extended form, suggesting that the formation of beta-sheet based fibrils does not require preformed extended structures. TTR 105-115Met111 displayed slight structural differences from TTR 105-115 which may be related to the fibril-forming propensity of the corresponding mutant TTR.

Homology model of thyroxine binding globulin and elucidation of the thyroid hormone binding site.

The tertiary structure of thyroxine binding globulin (TBG) has been modelled on the basis of its close homology to alpha 1-antitrypsin, the archetype of the serine protease inhibitor (serpin) superfamily. Energy minimization was applied to the model to refine the structure further. The putative thyroid hormone binding region suggested in previous labelling studies was found to exist within a beta-barrel structure of complementary dimensions to the thyroid hormones. The model also revealed that the binding cleft provides the hydrophobic environment and specific ionic interaction sites deemed important for thyroid hormone binding. The model is in good agreement with evidence derived from previously reported T3 and T4 binding, stability and isoelectric focussing studies of TBG and TBG variants. Finally, T4 analogue and drug binding studies have enabled us to postulate the orientation and manner of hormone binding to TBG. This may prove to be of assistance in the development of potent and specific, non-thyroidal ligands and also aid in the understanding of physiological thyroid hormone binding interactions.