Magic angle spinning (MAS) NMR: a new tool to study the spatial and electronic structure of photosynthetic complexes

Photosynthesis Research - In the last two decades, Magic Angle Spinning (MAS) NMR has created its own niche in studies involving photosynthetic membrane protein complexes, owing to its ability to...     

Magic angle spinning NMR spectroscopic metabolic profiling of gall bladder tissues for differentiating malignant from benign disease

Gall bladder tissue specimens obtained from 112 patients were examined by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Fifty one metabolites were identified by combination of one and two-dimensional NMR spectra. To our knowledge, this is the first report on metabolic profiling of gall bladder tissues using HR-MAS NMR spectroscopy. Metabolic profiles were evaluated for differentiation between benign Chronic Cholecystitis (CC, n = 66) and xantho-granulomatous cholecystitis (XGC, n = 21) and malignant gall bladder cancer (GBC, n = 25). Increase in choline containing compounds, amino acids, taurine, nucleotides and lactate as common metabolites were observed in malignant tissues whereas lipid content was found low as compared to benign tissues. Principal component analysis obtained from the NMR data showed clear distinction between CC and GBC tissue specimens; however, 27 % of XGC tissues were classified with GBC. The partial least square discriminant analysis (PLS-DA) multivariate analysis between benign (CC, XGC) and malignant (GBC) on the training data set (CC; n = 51, XGC; n = 15, GBC; n = 19 tissues specimens) provided 100 % sensitivity and 94.12 % specificity. This PLS-DA model when executed on the spectra of unknown tissue specimens (CC; n = 15, XGC; n = 6, GBC; n = 6) classified them into the three histological categories with more than 95 % of diagnostic accuracy. Non-invasive in vivo MRS technique may be used in future to differentiate between benign (CC and XGC) and malignant (GBC) gall bladder diseases.     

A high resolution (1)H magic angle spinning NMR study of a high-M(r) subunit of wheat glutenin

This work describes the application of (1)H magic angle spinning (MAS) nmr to the study of hydrated 1Dx5 wheat high-M(r) subunit. 1Dx5 is a water-insoluble 88 kDa protein, associated with good baking performance, and whose structure in the solid and low-hydration states is not known. High-resolution MAS (HR-MAS) results in a threefold resolution improvement of the (1)H spectra of the hydrated wheat protein, compared to standard MAS. The spectral resolution achieved enables, for the first time, two-dimensional nmr methods to be employed for the study of hydrated 1Dx5 and the assignment of the spectrum to be carried out on the basis of total correlated spectroscopy and (13)C/(1)H correlation experiments. Considerable shifts are observed for some resonances, relative to the chemical shifts of amino acids in solution, indicating that specific interactions occur in the hydrated protein network. Two main environments are identified for glutamine residues, Q(1) and Q(2), and these were characterized in terms of possible conformation and relative dynamics, with the basis of comparison between the single 90 degrees spectrum and the Carr-Purcel-Heiboom-Gill (CPMG) spectrum. The Q(1) residues are proposed to be situated in protein segments that adopt the beta-sheet conformation and that remain relatively hindered, possibly by hydrogen bonds involving the glutamine amide groups. On the other hand, Q(2) residues are proposed to be situated in a more mobile environment, adopting a looser conformation, possibly a beta-turn conformation. Based on the proximity of the Q(2) residues with glycine residues, as viewed by the nuclear Overhauser effect spectroscopy experiment, it is proposed that the protein segments that form the more mobile (or loop) sections of the network are rich in both glutamine and glycine residues.     

NMR solution structure and membrane interaction of the N-terminal sequence (1-30) of the bovine prion protein

The structure and membrane interaction of the N-terminal sequence (1-30) of the bovine prion protein (bPrPp) has been investigated by NMR spectroscopy in phospholipid membrane mimetic systems. CD spectroscopy revealed that the peptide adopts a largely alpha-helical structure in zwitterionic bicelles as well as in DHPC micelles but has a less degree of alpha-helix structure in partly charged bicelles. The solution structure of bPrPp was determined in DHPC micelles, and an alpha-helix was found between residues Ser8 and Ile21. The residues within the helical region show slow amide hydrogen exchange. Translational diffusion measurements in zwitterionic q = 0.5 bicelles show that the peptide does not induce aggregation of the bicelles. Increased quadrupolar splittings were observed in the outer part of the (2)H spectrum of DMPC in q = 3.5 bicelles, indicating that the peptide induces a certain degree of order in the bilayer. The amide hydrogen exchange and the (2)H NMR results are consistent with a slight positive hydrophobic mismatch and that bPrPp forms a stable helix that inserts in a transmembrane location in the bilayer. The structure of bPrPp and its position in the membrane may be relevant for the understanding of how the N-terminal (1-30) part of the bovine PrP functions as a cell-penetrating peptide. These findings may lead to a better understanding of how the prion protein accumulates at the membrane surface and also how the conversion into the scrapie form is carried out.     

Structure of Bombyx mori silk fibroin before spinning in solid state studied with wide angle x-ray scattering and (13)C cross-polarization/magic angle spinning NMR

The structure of a crystalline form of Bombyx mori silk fibroin, commonly found before the spinning process (known as silk I), has been proposed as a repeated beta-turn type II-like structure by combining data obtained from solid-state two dimensional spin-diffusion nuclear magnetic resonance and rotational-echo double-resonance (T. Asakura et al., J Mol Biol, in press). In this paper, the WAXS pattern of alanine-glycine alternating copolypeptide, (Ala-Gly)(15) with silk I form which was used for a silk I model of B. mori silk fibroin was observed. The pattern calculated with the silk I model proposed by us is well reproduced the observed one, indicating the validity of the proposed silk I model. In addition, two peptides of the other repeated sequences which contain Tyr or Val residues in the silk fibroin,23 were synthesized; (Ala-Gly-Tyr-Gly-Ala-Gly)(5) and (X-Gly)(15) where X is Tyr for the 7th, 15th and 23th residues, and Val for the 11th residue and Ala for other residues. There are no sharp peaks in the WAXS patterns, and therefore both samples are in the non-crystalline state. This is in agreement with the (13)C CP/MAS NMR result, where the conformation is mainly random coil.     

Static and magic angle spinning (31)P NMR spectroscopy of two natural plasma membranes

Static and magic angle spinning (31)P NMR spectroscopy was used for the first time in natural plasma membranes from erythrocytes and skeletal muscle to study phospholipid arrangement and composition. Typical static powder-like spectra were obtained showing that phospholipids were in a bilayer arrangement. Magic angle spinning narrowed spectra into two components. The first one corresponded to phosphatidylcholine and the second one to the other phospholipids with intensities in agreement with the known phospholipid composition. These findings show that NMR data previously acquired using model membranes can be transposed to studies on phospholipids in their natural environment.     

Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.     

NMR study on Pt(II) interaction with Amadori compounds

Here we report the first evidence of Pt(II) interaction with Amadori compound [N-(1-deoxy-d-Fructos-1-yl)glycine (Fru-Hgly)]. The ¹H and ¹⁹⁵Pt NMR results show that complexation of Pt(II) by Fru-Hgly is strongly dependent on pH and reaction molar ratio. In 1/1 Pt/Fru-Hgly molar ratio, at acidic pH, the first coordination site is the carboxylic oxygen, while at physiological pH the anchoring group is the aminic one, in both cases the system slowly evolves towards an N,O chelating mode. In 1/2 Pt/Fru-Hgly molar ratio the only coordination site is nitrogen atom while the carboxylic oxygen is not involved in metal coordination.     

Magic angle sample spinning 13C nuclear magnetic resonance of isotopically labeled bacteriorhodopsin

Bacteriorhodopsin (bR), the light-driven proton pump protein from Halobacterium halobium, was biosynthetically labeled with [4-13C]Asp. The incorporation yield was 48%. The magic angle sample spinning (MASS) 13C nuclear magnetic resonance (NMR) spectrum of this sample revealed six different peaks superimposed on a broad band of naturally abundant peptide-bond 13C. Two of the six carbonyl signals can be attributed to internal-protonated Asp carboxyl groups, one of which might be Asp115. An additional resonance at 110 ppm can be associated with the C-11 carbon of Trp, indicating an unusual biosynthetic pathway of this amino acid in Halobacterium halobium. Similar measurements performed on papain-treated purple membrane which lacks the C-terminal tail display two new intense signals at 178 and 178.9 ppm. If the same spectrum is taken without cross-polarization, these signals do not decrease or disappear. On the basis of their intensities and their chemical shifts, one can assign in addition to the C-terminal Asp four Asp residues facing the cytoplasmic phase. In native bR, at least two of these form a salt-bridge-like bond which also might include the C-terminal tail. These experiments not only provide data about the chemical environment of the Asp residues within the hydrophobic core of bacteriorhodopsin but also yield information about the interactions between surface components.     

NMR solution structure and SRP54M predicted interaction of the N-terminal sequence (1-30) of the ovine Doppel protein

Prion protein (PrP^C) biosynthesis involves a multi-step process that includes translation and post-translational modifications. While PrP has been widely investigated, for the homolog Doppel (Dpl), limited knowledge is available. In this study, we focused on a vital step of eukaryotic protein biosynthesis: targeting by the signal recognition particle (SRP). Taking the ovine Dpl (OvDpl(1-30)) peptide as a template, we studied its behavior in two different hydrophobic environments using CD and NMR spectroscopy. In both trifluoroethanol (TFE) and dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC), the OvDpl(1-30) peptide revealed to fold in an alpha-helical conformation with a well-defined central region extending from residue Cys8 until Ser22. The NMR structure was subsequently included in a computational docking complex with the conserved M-domain of SRP54 protein (SRP54M), and further compared with the N-terminal structures of mouse Dpl and bovine PrP^C proteins. This allowed the determination of (i) common predicted N-terminal/SRP54M polar contacts (Asp331, Gln335, Glu365 and Lys432) and (ii) different N–C orientations between prion and Dpl peptides at the SRP54M hydrophobic groove, that are in agreement with each peptide electrostatic potential. Together, these findings provide new insights into the biosynthesis of prion-like proteins. Besides they also show the role of protein conformational switches in signalization toward the endoplasmic membrane, a key event of major significance in the cell cycle. They are thus of general applicability to the study of the biological function of prion-like as well as other proteins.     

Magic angle spinning NMR-based metabolic profiling of head and neck squamous cell carcinoma tissues

High-resolution magic-angle spinning (HR-MAS) proton NMR spectroscopy is used to explore the metabolic signatures of head and neck squamous cell carcinoma (HNSCC) which included matched normal adjacent tissue (NAT) and tumor originating from tongue, lip, larynx and oral cavity, and associated lymph-node metastatic (LN-Met) tissues. A total of 43 tissues (18 NAT, 18 Tumor and 7 LN-Met) from 22 HNSCC patients were analyzed. Principal Component Analysis of NMR data showed a clear classification between NAT and tumor tissues, however, LN-Met tissues were classified among tumor. A partial least-squares discriminant analysis model generated from NMR metabolic profiles was used to differentiate normal from tumor samples (Q(2) > 0.80, Receiver Operator Characteristic area under the curve >0.86, using 7-fold cross validation). HNSCC and LN-Met tissues showed elevated levels of lactate, amino acids including leucine, isoleucine, valine, alanine, glutamine, glutamate, aspartate, glycine, phenylalanine and tyrosine, choline containing compounds, creatine, taurine, glutathione, and decreased levels of triglycerides. These elevated metabolites were associated with highly active glycolysis, increased amino acids influx (anaplerosis) into the TCA cycle, altered energy metabolism, membrane choline phospholipid metabolism, and oxidative and osmotic defense mechanisms. Moreover, decreased levels of triglycerides may indicate lipolysis followed by β-oxidation of fatty acids that may exist to deliver bioenergy for rapid tumor cell proliferation and growth.     

Molecular orientation and conformation of phosphatidylinositides in membrane mimetics using variable angle sample spinning (VASS) NMR

For many biological molecules, determining their geometry as they exist in a membrane environment is a crucial step in understanding their function. Variable angle sample spinning (VASS) NMR provides a new route to obtaining geometry information on membrane-associating molecules; it has been used here to scale and separate anisotropic contributions to phosphorus chemical shifts in NMR spectra of phosphatidylinositol phosphates. The procedure allows spectral assignment via correlation with isotropic chemical shifts and determination of a family of probable headgroup orientations via interpretation of anisotropic shift contributions. The molecules studied include phosphtidylinositol-4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). A membrane-like environment is provided by a dispersion of alkyl-poly(ethylene) glycols and n-alcohols that forms a field-orienting liquid crystal with a director that can be manipulated by varying the sample spinning axis. The experiments presented indicate that the variable angle sample spinning method will provide a direct approach for assignment and extraction of structural information from membrane-associating biomolecules labeled with a wider variety of NMR active isotopes.     

Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state (13)C-NMR and differential scanning calorimetry

Phosphatidylserine (PS) extracted from pig brain and synthetic dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were used to make DPPC/DMPC and DPPC/PS large unilamellar liposomes with a diameter of approximately 1 microm. Chlorpromazine-HCl (CPZ), an amphipathic cationic psychotropic drug of the phenothiazine group, is known to partition into lipid bilayer membranes of liposomes with partition coefficients depending on the acyl chain length and to alter the bilayer structure in a manner depending on the phospholipid headgroups. The effects of adding CPZ to these membranes were studied by differential scanning calorimetry and proton cross polarization solid state magic angle spinning (13)C-nuclear magnetic resonance spectroscopy (CP-MAS-(13)C-NMR). CP-MAS-(13)C-NMR spectra of the DPPC (60%)/DMPC (40%) and the DPPC (54%)/DMPC (36%)/CPZ (10%) liposomes, show that CPZ has low or no interaction with the phospholipids of this neutral and densely packed bilayer. Conversely, the DPPC (54%)/PS (36%)/CPZ (10%) bilayer at 25 degrees C demonstrates interaction of CPZ with the phospholipid headgroups (PS). This CPZ interaction causes about 30% of the acyl chains to enter the gauche conformation with low or no CPZ interdigitation among the acyl chains at this temperature (25 degrees C). The DPPC (54%)/PS (36%)/CPZ (10%) bilayer at a sample temperature of 37 degrees C (T(C)=31.2 degrees C), shows CPZ interdigitation among the phospholipids as deduced from the finding that approximately 30% of the phospholipid acyl chains carbon resonances shift low-field by 5-15 ppm.     

Investigating the interaction of saposin C with POPS and POPC phospholipids: a solid-state NMR spectroscopic study

The interaction of Saposin C (Sap C) with negatively charged phospholipids such as phosphatidylserine (PS) is essential for its biological function. In this study, Sap C (initially protonated in a weak acid) was inserted into multilamellar vesicles (MLVs) consisting of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (negatively charged, POPS) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (neutrally charged, POPC). The MLVs were then investigated using solid-state NMR spectroscopy under neutral pH (7.0) conditions. The (2)H and (31)P solid-state NMR spectroscopic data of Sap C-POPS and Sap C-POPC MLVs (prepared under the same conditions) were compared using the (2)H order parameter profiles of the POPC-d(31) or POPS-d(31) acyl chains as well as the (31)P chemical shift anisotropy width and (31)P T(1) relaxation times of the phospholipids headgroups. All those solid-state NMR spectroscopic approaches indicate that protonated Sap C disturbs the POPS bilayers and not the POPC lipid bilayers. These observations suggest for the first time that protonated Sap C inserts into PS bilayers and forms a stable complex with the lipids even after resuspension under neutral buffer conditions. Additionally, (31)P solid-state NMR spectroscopic studies of mechanically oriented phospholipids on glass plates were conducted and perturbation effect of Sap C on both POPS and POPC bilayers was compared. Unlike POPC bilayers, the data indicates that protonated Sap C (initially protonated in a weak acid) was unable to produce well-oriented POPS bilayers on glass plates at neutral pH. Conversely, unprotonated Sap C (initially dissolved in a neutral buffer) did not interact significantly with POPS phospholipids allowing them to produce well-oriented bilayers at neutral pH.     

Monitoring of the ethionamide pro-drug activation in mycobacteria by (1)H high resolution magic angle spinning NMR

In this study, we use HRMAS NMR as a non-invasive technique to monitor the in vivo metabolism of a xenobiotic. The antituberculosis Ethionamide is a pro-drug that has to be activated in mycobacteria before inhibiting its cellular target. The use of (1)H HRMAS NMR has allowed to detect a metabolite (ETH*) of the drug directly in living bacteria, even with a spectrometer operating at the relatively low magnetic field of 300MHz. We show that metabolism monitoring of an unlabelled drug at a therapeutically relevant concentration as low as 5mug/ml is within reach of the technique. (1)H HRMAS NMR in combination with diffusion filtering leads to the conclusion that the metabolite is located inside the intact cells. The comparison of the metabolite NMR signature with that of synthetic molecules proves the non-identity of ETH* with the ETH derivatives described previously in the literature.     

Reactivity of Al(III) with membrane phospholipids: a NMR approach

The complexes Al(acac)₃ (1) (acac = 2,4-pentanedionate) and Al(malt)₃ (malt = 3-hydroxy-2-methyl-4-pyronate) (2) react with dl--dipalmitoylphosphatidylcholine (DPPC) under a 1:1 molar ratio in CDCl₃ at 37 °C, as shown by the substantial release of ligands (20–50%) from the metal coordination sphere (¹H-NMR), by evident changes in the ¹H-NMR spectrum of DPPC in the reaction mixture and by the appearance of a ³¹P-NMR signal due to metal-coordinated DPPC. ³¹P-NMR spectra reveal that both 1 and 2 also react with DPPC in water, in the presence of 1% Triton X-100 and Tris buffer. Under these conditions, 1 and 2 do not react with ghosts from human erythrocytes. On the contrary, the far less hydrolytically stable complex Al(lact)₃ (lact = lactate) appears to be reactive under identical conditions, as shown by ³¹P-NMR spectra.     

A (1)H NMR study of the interaction of antitumor metallocenes with glutathione

The interaction of the antitumor active metallocene dihalides Cp(2)MCl(2) (M=Ti, Nb, Mo) and 1 equiv. of glutathione was studied by (1)H NMR spectroscopy at pD 2-7 in 4 mM NaCl solutions. No interaction between glutathione and titanocene dichloride was detected at pD 2, while at pD 5-7 competitive hydrolysis of the cyclopentadienyl ligands occurred. With niobocene dichloride formation of approximately 20% of an adduct was observed at pD 2 and 5, but hydrolysis of the Cp ligands in the adduct occurred over 24 h. Molybdocene dichloride formed two stable adducts at pD 6 which were tentatively assigned as a Cp(2)Mo-glutathione chelate involving coordination of the cysteine thiol and glycine carboxylate to the metal centre, and a thiol centred 1:2 Cp(2)Mo-glutathione complex. The implications for the mechanism of antitumor action of the metallocene dihalides is discussed.     

Chlorpromazine interaction with phosphatidylserines: a (13)C and (31)P solid-state NMR study

Niemann-Pick type C, or NPC for short, is an early childhood disease exhibiting progressive neurological degeneration, associated with hepatosplenomegaly in some cases. The disease, at the cellular level, is a result of improper trafficking of lipids such as cholesterol and glycosphingolipids (GSLs) to lysosome-like storage organelles (LSOs), which become engorged with these lipids. It is believed that the initial defect in trafficking, whether of cholesterol or a GSL, results in an eventual traffic jam in these LSOs. This leads to the retention of not only other lipids, but also of transmembrane proteins that transiently associate with the late endosomes (LE) in normal cells, on their way to other cellular destinations such as the trans-Golgi network (TGN). In this review, we discuss the biophysical properties of lipids and cholesterol that might determine their intracellular itineraries, and how these itineraries are altered in NPC cells, which have defects in the proteins NPC1 or NPC2. We also discuss some potential therapeutic directions being suggested by recent research.     

Solution structure of the two N-terminal RNA-binding domains of nucleolin and NMR study of the interaction with its RNA target

Nucleolin is an abundant 70 kDa nucleolar protein involved in many aspects of ribosomal RNA biogenesis. The central region of nucleolin contains four tandem consensus RNA-binding domains (RBD). The two most N-terminal domains (RBD12) bind with nanomolar affinity to an RNA stem-loop containing the consensus sequence UCCCGA in the loop. We have determined the solution structure of nucleolin RBD12 in its free form and have studied its interaction with a 22 nt RNA stem-loop using multidimensional NMR spectroscopy. The two RBDs adopt the expected beta alpha beta beta alpha beta fold, but the position of the beta 2 strand in both domains differs from what was predicted from sequence alignments. RBD1 and RBD2 are significantly different from each others and this is likely important in their sequence specific recognition of the RNA. RBD1 has a longer alpha-helix 1 and a shorter beta 2-beta 3 loop than RBD2, and differs from most other RBDs in these respects. The two RBDs are separated by a 12 amino acid flexible linker and do not interact with one another in the free protein. This linker becomes ordered when RBD12 binds to the RNA. Analysis of the observed NOEs between the protein and the RNA indicates that both RBDs interact with the RNA loop via their beta-sheet. Each domain binds residues on one side of the loop; specifically, RBD2 contacts the 5' side and RBD1 contacts the 3'.