Solid-state NMR studies of 1H spin diffusion in adsorbed organic molecules

1H spin diffusion times of toluene (MB) and tetrahydrofuran (THF) adsorbed on a series of porous solids (charcoal, SiO2 and Al2O3) were measured by a selective inversion technique. The experimental results show that they cover a wide range (from less than one millisecond to several hundreds of milliseconds). For all samples, a tri-exponential behavior was observed in the magnetization recovery processes of the negative peaks. This is attributed to the existence of the two different kinds of spin diffusion processes in addition to the T1 relaxation. One is assigned to the intermolecular spin diffusion between the surface acidic protons of the adsorbent and the organic molecules adsorbed on the solid surface, the other to the intramolecular spin diffusion of adsorbed molecules. Due to hydrogen bonding between the surface hydroxyl groups and the adsorbate, the intermolecular spin diffusion of THF adsorbed on various solids is more effective compared to that of adsorbed MB. In addition, the intermolecular 1H spin diffusion between charcoal and adsorbed THF molecules was confirmed by indirect measurement suggested by Tekely et al.     

Solid-state NMR studies of proteins: the view from static 2H NMR experiments

The application of solid-state 2H NMR spectroscopy to the study of protein and peptide structure and dynamics is reviewed. The advantages of solid-state NMR for the study of proteins are considered, and the particular advantages of solid-state 2H NMR are summarized. Examples of work on the integral membrane protein bacteriorhodopsin, and the membrane peptide gramicidin, are used to highlight the major achievements of the 2H NMR technique. These examples demonstrate that through the use of oriented samples, it is possible to obtain both structural and dynamic information simultaneously.     

Solid-state NMR studies of magnetically aligned phospholipid membranes: taming lanthanides for membrane protein studies

The addition of lanthanides (Tm3+, Yb3+, Er3+, or Eu3+) to a solution of long-chain phospholipids such as dimyristoylphosphatidylcholine (DMPC) and short-chain phospholipids such as dihexanoylphosphatidylcholine (DHPC) is known to result in a bilayer phase in which the average bilayer normal aligns parallel to an applied magnetic field. Lanthanide-doped bilayers have enormous potential for the study of membrane proteins by solid-state NMR, low-angle diffraction, and a variety of optical spectroscopic techniques. However, the addition of lanthanides poses certain challenges to the NMR spectroscopist: coexistence of an isotropic phase and hysteresis effects, direct binding of the paramagnetic ion to the peptide or protein of interest, and severe paramagnetic shifts and line broadening. Lower water concentrations and larger DMPC/DHPC ratios than those typically used in bicelles consistently yield a single oriented bilayer phase that is stable over a wide range of temperature (approximately 35-90 degrees C). Among the above choice of lanthanides, Yb3+ is found to give minimal paramagnetic shifts and line broadening at acceptably low concentrations necessary for alignment (i.e., Yb3+/DMPC mole ratios equal to or greater than 0.01). Finally, the addition of a phospholipid chelate, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine--diethylenetriaminepent aacetic acid, is observed to significantly reduce paramagnetic broadening and presumably prevent direct association of the peptide with the lanthanide ions.     

A prion protein fragment primes type 1 astrocytes to proliferation signals from microglia

Giliosis is a hallmark of prion disease. A neurotoxic prion peptide (PrP106-126) induces astrocyte proliferation in the presence of microglia. This peptide also directly enhances microglial proliferation in culture. We have investigated this further to understand the method by which factors released by microglia and PrP106-126 work together to enhance astrocyte proliferation. PrP106-126 in the presence of microglia specifically enhanced type 1 astrocyte proliferation but not Type 2. Astrocytes that do not express the prion protein were more sensitive to oxidative stress and the toxicity of cytosine arabinoside. In the presence of cytosine arabinoside, PrP106-126 was toxic to pure astrocyte cultures. Using conditioned medium from microglia we have shown that PrPc-expressing astrocytes proliferate in response to factors released by microglia stimulated by granulocyte/macrophage colony-stimulating factor. This response is enhanced in the presence of PrP106-126. PrPc-deficient astrocytes do not show this response. These results suggest that astrocytes are primed by PrP106-126 to respond more to factors released by proliferating microglia. Astrocytes may proliferate in this system to escape entering the cell suicide pathway.     

Solid-state 2H NMR study of methyl-d3-cobalamin

A solid-state 2H NMR study of methyl-d3-cobalamin has been performed as a function of temperature to provide information concerning the character and energetics of the motion performed by this unique bioorganometallic methyl group. Analysis of the 2H NMR line shape indicates that the methyl group undergoes rapid three-fold rotation, and that the Co-C-2H angle lies between 105.9 and 109.5 degrees. Determination of the spin-lattice relaxation times T1 shows that the relaxation is anisotropic, consistent with a "jumping" motion of the methyl group rather than rotational diffusion. This also provides the activation energy to methyl jumps as 8.3 +/- 1.3 kJ/mol. It is proposed that this energetic barrier may be a useful probe of changes in the electronic character of the Co-C bond that accompany the biological role of this molecule in such enzymes as methionine synthase.     

Interactions of a vesicular stomatitis virus G protein fragment with phosphatidylserine: NMR and fluorescence studies

Closed-loop recycling with periodic intra-profile injection (CLRPIPI) is a binary chromatographic separation technique. It is similar to simulated moving bed chromatography in that sample is injected into the interior of the circulating chromatographic profile. Although the CLRPIPI process is repetitive, it is different from SMB in that CLRPIPI is not continuous. It is shown that the CLRPIPI process reaches a steady state and that high purity fractions can be collected.     

Two-dimensional 1H and 15N NMR titration studies of hisactophilin

We have used two-dimensional 1H-15N heteronuclear single quantum correlation spectroscopy to measure the pH dependence of backbone amide group chemical shifts in the actin binding protein hisactophilin over the pH range 5.7-11.1. Most of the resonances can be analyzed using a simple equation involving a single apparent ionization constant, pK(app). The majority of resonances in the protein titrate with pK(app) values of 5.6-7.4. The results can be rationalized in terms of titration of many histidine residues in hisactophilin. The titration data provide direct experimental support for the proposed models of the atomic basis of actin and membrane binding by hisactophilin.     

1H and 13C NMR studies of conformational behaviour of Leu-enkephalin

The presence of secondary sensory cells in the Octopus gravity receptor system has been demonstrated. In serial thin sections of the receptor cells (hair cells) no axons were found leaving the cells. Instead, synapses were observed with synaptic vesicles lying inside the receptor cells. Both data clearly indicate that the receptor hair cells represent secondary sensory cells. In addition, efferent contacts to the receptor cells could be confirmed.     

Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR

Raphanus sativus Antifungal Protein 1 (Rs-AFP1) is a 51 amino acid residue plant defensin isolated from radish (Raphanus sativus L.) seeds. The three-dimensional structure in aqueous solution has been determined from two-dimensional 1H NMR data recorded at 500 MHz using the DIANA/REDAC calculation protocols. Experimental constraints consisted of 787 interproton distances extracted from NOE cross-peaks, 89 torsional constraints from 106 vicinal interproton coupling constants and 32 stereospecific assignments of prochiral protons. Further refinement by simulated annealing resulted in a set of 20 structures having pairwise root-mean-square differences of 1.35(+/- 0.35) A over the backbone heavy atoms and 2.11(+/- 0.46) A over all heavy atoms. The molecule adopts a compact globular fold comprising an alpha-helix from Asn18 till Leu28 and a triple-stranded beta-sheet (beta 1 = Lys2-Arg6, beta 2 = His33-Tyr38 and beta 3 = His43-Pro50). The central strand of this beta-sheet is connected by two disulfide bridges (Cys21-Cys45 and Cys25-Cys47) to the alpha-helix. The connection between beta-strand 2 and 3 is formed by a type VIa beta-turn. Even the loop (Pro7 to Asn17) between beta-strand 1 and the alpha-helix is relatively well defined. The structure of Raphanus sativus Antifungal Protein 1 features all the characteristics of the "cysteine stabilized alpha beta motif". A comparison of the complete structure and of the regions important for interaction with the fungal receptor according to a mutational study, is made with the structure of gamma-thionin, a plant defensin that has no antifungal activity. It is concluded that this interaction is both electrostatic and specific, and some possible scenarios for the mode of action are given.     

1H NMR studies of the bis-intercalation of a homodimeric oxazole yellow dye in DNA oligonucleotides

Retention of apo B-100 lipoproteins, low density lipoprotein (LDL) and probably lipoprotein(a), Lp(a), by intima proteoglycans (PGs) appears to increase the residence time needed for their structural, hydrolytic and oxidative modifications. If the rate of LDL entry exceeds the tissue capacity to eliminate the modified products, this process may be a contributor to atherogenesis and lesion advancement. LDL binds to PGs of the intima, by association of specific positive segments of the apo B-100 with the negatively-charged glycosaminoglycans (GAGs) made of chondroitin sulfate (CS), dermatan sulfate (DS) and probably heparan sulfate (HS). Small, dense LDL has a higher affinity for CS-PGs than large buoyant particles, probably because they expose more of the segments binding the GAGs than larger LDL. PGs cause irreversible structural alterations of LDL that potentiate hydrolytic and oxidative modifications. These alterations also increase LDL uptake by macrophages and smooth muscle cells. These in vitro data suggest that part of the atherogenicity of LDL may depend on its tendency to form complexes with arterial PGs in vivo. Ex vivo results support this hypothesis. Subjects with coronary heart disease have LDL with significantly higher affinity for arterial PGs. This is also a characteristic of subjects with the atherogenic lipoprotein phenotype, with high levels of small, dense LDL. The LDL-PG affinity, however can be modified by dietary or pharmacological interventions that change the composition and size of LDL. Lesion-prone intima contain PGs with a high affinity for LDL. Increased LDL entrapment at these sites may be a key step in a cyclic atherogenic process.     

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of approximately 142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an alpha-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular beta-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.     

1H NMR spectroscopy studies of Huntington's disease: correlations with CAG repeat numbers

Huntington's disease (HD) is the result of an expanded (CAG) repeat in a gene on chromosome 4. A consequence of the gene defect may be progressive impairment of energy metabolism. We previously showed increased occipital cortex lactate in HD using localized 1H spectroscopy. We have now extended these studies to show an almost threefold elevation in occipital cortex lactate in 31 HD patients as compared with 17 normal control subjects (p < 10(-11)). The spectra in three presymptomatic gene-positive patients were identical to normal control subjects in cortical regions, but three in eight showed elevated lactate in the striatum. Similar to recently reported increases in task-related activation of the striatum in the dominant hemisphere, we found that striatal lactate levels in HD patients were markedly asymmetric (higher on the left side). Markers of neuronal degeneration, decreased N-acetylaspartate (NAA)/creatine and increased choline/creatine levels, were symmetric. Both decreased NAA and increased lactate in the striatum significantly correlated with duration of symptoms. When divided by his or her age, an individual's striatal NAA loss and lactate increase were found to directly correlate with the subject's CAG repeat number, with correlation coefficients of 0.8 and 0.7, respectively. Similar correlations were noted between postmortem cell loss and age versus CAG repeat length. Together, these data provide further evidence for an interaction between neuronal activation and a defect in energy metabolism in HD that may extend to presymptomatic subjects.     

1H NMR structure of an antifungal gamma-thionin protein SIalpha1: similarity to scorpion toxins

The three-dimensional structure of the Sorghum bicolor seed protein gamma-thionin SIalpha1 has been determined by 2D 1H nuclear magnetic resonance (NMR) spectroscopy. The secondary structure of this 47-residue antifungal protein with four disulphide bridges consists of a three-stranded antiparallel sheet and one helix. The helix is tethered to the sheet by two disulphide bridges which link two successive turns of the helix to alternate residues i, i+2 in one strand. Possible binding sites for antifungal activity are discussed. The same fold has been observed previously in several scorpion toxins.     

1H/27Al TRAPDOR NMR studies on aluminum species in dealuminated zeolites

Aluminum species in several dealuminated zeolites (ultrastable HY, HZSM-5 and mordenite) were investigated in detail by means of the newly introduced 1H/27Al TRAPDOR method in combination with 27Al MAS NMR, and the quadrupole coupling constants (Q[CC]s) for aluminum atoms associated with these species were obtained. A signal at ca. 6.8 ppm, due to water molecules adsorbed on Lewis acid sites, was observed in the 1H MAS spectra for all the three zeolites. The TRAPDOR NMR provides direct evidence that there is a strong interaction between the adsorbed water molecules and the aluminum atoms of the Lewis-acid sites. The Q(CC) values for this aluminum species of 8.3, 6.7 and 11.3 MHz were determined from the TRAPDOR profiles for the ultrastable HY, HZSM-5 and mordenite zeolites, respectively. The Q(CC)s calculated from the TRAPDOR curves are usually larger than 10 MHz for both Bronsted-acid sites (SiOHAI) and non-framework aluminum species in the three zeolites. Three narrow peaks at 54, 30 and 0 ppm are separately superimposed on a broad hump in the 27Al MAS spectra of the three dehydrated zeolites, while the latter is associated with the 'NMR invisible' Al. The NMR experimental results suggest that the three kinds of aluminum species (non-framework aluminum species, Bronsted- and Lewis-acid sites) are all responsible for the resonance of the broad hump in dehydrated zeolites, which makes it difficult to explain the 27Al MAS spectra. Fortunately, the TRAPDOR NMR provides a direct method for individually studying different aluminum species with large Q(CC)s via their dipolar coupling to nearby proton nuclei.     

Propagation of prion strains through specific conformers of the prion protein

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.     

1H MAS and 1H[23Na] double resonance NMR studies on the modification of surface hydroxyl groups of gamma-alumina by sodium

The modification of surface hydroxyl groups with sodium in a series of Na2CO3-gamma-Al2O3 catalysts was investigated as a function of both the Na2CO3 loading and the calcination temperature by means of 1H magic angle spinning (MAS) and 1H(23Na) spin-echo double resonance NMR techniques. The 1H NMR experiments revealed that sodium ions are homogeneously distributed over the alumina surface and closely coordinated with the surface hydroxyl groups. In the catalysts calcined at 250 degrees C, the acidic hydroxyl groups (with a chemical shift of 2.0 ppm) are preferentially associated with sodium ions at low Na2CO3 coverages (5 and 10%), while both the acidic and the basic (0 ppm) hydroxyl groups are accessible for sodium ions at high coverages (15 and 20%). The coordination causes a low-field shift of about 2 ppm in the 1H MAS spectra, and a broad signal at 4.5 ppm appears. It is interesting that the 4.5 ppm signal is completely suppressed in the 1H(23Na) MAS experiments, providing direct evidence that a strong interaction exists between adsorbed sodium ions and the surface hydroxyl groups. Increasing the calcination temperature to 450 degrees C results in preferential removal of the acidic hydroxyl groups, and only the most basic hydroxyl groups remain when the calcination temperature is raised to 600 degrees C. This is attributed to the formation of the coordinated species. [formula: see text] which enhances the acidity of the surface hydroxyl groups and prompts their dehydroxylation, especially at high calcination temperature. Correlation of the 1H MAS NMR results and catalytic activity measurements indicates that the basic hydroxyl groups are essential for the carbonyl sulfide hydrolysis reaction.     

Ultrastructural studies on scrapie prion protein crystals obtained from reverse micellar solutions

The structural transition from the cellular prion protein (PrPC) that is rich in alpha-helices to the pathological form (PrPSc) that has a high beta-sheet content seems to be the fundamental event underlying the prion diseases. Determination of the structure of PrPSc and the N-terminally truncated PrP 27-30 has been complicated by their insolubility. Here we report the solubilization of PrP 27-30 through a system of reverse micelles that yields monomeric and dimeric PrP. Although solubilization of PrP 27-30 was not accompanied by any recognizable change in secondary structure as measured by FTIR spectroscopy, it did result in a loss of prion infectivity. The formation of small two- and three-dimensional crystals upon exposure to uranyl salts argues that soluble PrP 27-30 possesses considerable tertiary structure. The crystals of PrP 27-30 grown from reverse micellar solutions suggest a novel crystallization mechanism that might be applicable for other membrane proteins. A variety of different crystal lattices diffracted up to 1.85 nm by electron microscopy. Despite the lack of measurable biological activity, the structure of PrP 27-30 in these crystals may provide insight into the structural transition that occurs during PrPSc formation.     

Measurement of molecular association in drug: cyclodextrin inclusion complexes with improved 1H NMR studies

The molecular association of haloperidol with hydroxypropyl-beta-cyclodextrin, expressed by the binding constant of the inclusion complex formed, was calculated from the changes on the 1H NMR spectra of the drug in the presence of the cyclodextrin. The stoichiometry of the complex was calculated by use of the continuous variation method (Job plot), and found to be 1:1. The binding constant for the 1:1 complex was calculated using improved non-linear models, which were solved by non-linear least-squares regression analysis, applying an iteration procedure. Three improved mathematical models for more accurate calculation of binding constants are proposed. The models are free from any assumptions and from practical or theoretical shortcomings.     

Proof of the primacy of prion protein

This paper presents a case of a 5-year old girl with a congenital absence of the nose. Congenital arrhinia is a very rare malformation of the midfacial bones. The difficulties of treating a child with this abnormality are discussed.