Structural Characterization of Polyglutamine Fibrils by Solid-State NMR Spectroscopy

Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington's disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely β-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for β-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended β-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing β-strands to more than one sheet and an antiparallel assembly of strands within β-sheets.     

环化二磷酸腺苷核糖及其钙动员的功能

环化二磷酸腺苷核糖（cyclic ADP-ribose,cADPR）是烟酰胺腺嘌呤二核苷酸(NAD⁺)的代谢产物,是新近发现的一种细胞内第二信使．在许多哺乳类和无脊椎动物细胞中,cADPR能引起胞内钙库释放钙离子,其可能机制是：cADPR受体结合cADPR,通过Ryanodine受体或类Ryanodine受体介导的钙通道使cADPR敏感的钙库释放钙离子,此外,一条由一氧化氮（NO）、环化鸟苷酸（cGMP）和cADPR组成的细胞内信号转导途径可能存在于许多细胞中．     

Molecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolase

Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys²⁵³), either within the metallo-dependent phosphatases signature (Gln²⁷, Asn¹¹⁰, His¹¹¹), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe³⁷ and Arg⁴³) and h7h8 (Phe²¹⁰), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe³⁷ was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose K_m and unchanged k_cat of F37A-ADPRibase-Mn, while the K_m values for the other substrates were little affected. Arg⁴³ was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys²⁵³ was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´10⁴ M^-1s^-1 versus 4´10³ M^-1s^-1 of the wild type.     

Analysis of Structural Variability in Pharmaceutical Excipients Using Solid-State NMR Spectroscopy

Polysaccharide-based excipients comprise the majority of most solid dosage forms and can vary dramatically in terms of structural and functionally related properties. Analytical methods for characterizing these important formulation components are crucial. Solid-state NMR spectroscopy (SSNMR) can provide a wealth of information on these materials while offering the advantages of non-destructive sample preparation and selectivity. The overall objective of this work is to identify SSNMR parameters that can be used to detect differences among these excipients. Excipients were obtained from a wide range of suppliers and analyzed as received; ¹³C SSNMR spectra were acquired using a Chemagnetics CMX-300 spectrometer operating at approximately 75 MHz. The resolution of SSNMR signals of many excipients allows for positive identification of the major form present. Alginic acid and sodium alginate can be differentiated based on carbonyl peak position. Analysis of relative peak intensities provides insight into the purity of a carrageenan sample compared to known standards. The SSNMR spectrum of starch can be used to identify the source and to quantitate the amorphous and crystalline content. Relaxation values and peak areas of starch derivatives can be related to the degree of hydrolysis, providing an alternative method for determining dextrose equivalent. Differences in peak intensities and relaxation time values of HPMC samples can be correlated to the amount of methoxy subsituent groups. Important characteristics of excipients such as form identification, structural differences, crystalline and amorphous content, and water content variations can be detected using SSNMR spectroscopy.     

Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-¹³C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-¹³C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the ¹³C NMR spectra due to loss of the label as ¹³CO₂. Uptake of [3-¹³C]pyruvate by the cells was considerably slower than with [2-¹³C]acetate; however, the labelling patterns were similar with the exception of increased [3-¹³C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-¹³C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine.     

Structural proteomics by NMR spectroscopy

Structural proteomics is one of the powerful research areas in the postgenomic era, elucidating structure-function relationships of uncharacterized gene products based on the 3D protein structure. It proposes biochemical and cellular functions of unannotated proteins and thereby identifies potential drug design and protein engineering targets. Recently, a number of pioneering groups in structural proteomics research have achieved proof of structural proteomic theory by predicting the 3D structures of hypothetical proteins that successfully identified the biological functions of those proteins. The pioneering groups made use of a number of techniques, including NMR spectroscopy, which has been applied successfully to structural proteomics studies over the past 10 years. In addition, advances in hardware design, data acquisition methods, sample preparation and automation of data analysis have been developed and successfully applied to high-throughput structure determination techniques. These efforts ensure that NMR spectroscopy will become an important methodology for performing structural proteomics research on a genomic scale. NMR-based structural proteomics together with x-ray crystallography will provide a comprehensive structural database to predict the basic biological functions of hypothetical proteins identified by the genome projects.     

Structural Determination of Silicon-Containing Oligonucleotides by 1H−29Si Long-Range Heteronuclear Multiple Quantum Correlation NMR Spectroscopy

Abstract

The technique of ¹H^?29Si Long-Range Heteronuclear Multiple Quantum Correlation NMR Spectroscopy was used to determine the structure of silicon-containing oligonucleotides. Trimers which contained silicon instead of phosphorus as part of the oligonucleotide link were synthesized through a synthetic route that required minimal hydroxyl protection. The resulting trimer could have one of two possible structures. Through the use of ¹H^?29Si HMQC NMR spectroscopy, it was possible to link the 3′-hydroxymethine proton of one sugar to the 5′-hydroxymethylene proton of an adjacent sugar by correlation to the same silicon atom, thus elucidating the final structure.     

低能铁离子束辐照氨基酸衍生物的红外光谱结构表征

利用傅里叶变换红外光谱（FTIR）技术研究了能量为110keV的Fe^ 离子束辐照L（ ）-半胱氨酸盐酸盐单晶水合物固态样品的结构变化情况。对辐照样品的两次红外光谱分析结果有所不同,表明固态样品不同部位受低能铁离子束作用后可发生不同的分子改性;但二者又具有一定的相似性,都反映了原分子中氨基、羧基和巯基等基团的受损及硝基和酰胺基团等的生成。     

Structural analysis of galactomannans by NMR spectroscopy

The structure of naturally occurring galactomannans was characterized by high resolution NMR spectroscopy involving two-dimensional (2D) NMR measurements of the field gradient DQF-COSY, HMQC, HMBC, and ROESY experiments. Four galactomannans with different proportions of galactose (G) and mannose (M), from fenugreek gum (FG), guar gum (GG), tara gum (TG), and locust bean gum (LG), were investigated. Because these galactomannans had very high molecular weights, hydrolysis by dilute H₂SO₄ was carried out to give the corresponding low molecular weight galactomannans, the structural identities of which were established by comparison of the specific rotations, shape of the GPC profiles, and NMR spectra with those of higher molecular weight galactomannans. The correlation signals GH1-GC4, -GC5, and -MC6 in HMBC and GH1-GH6 in ROESY spectra of FG showed that more than two galactopyranose units with the 1 → 4 linkage were connected at C6 of the mannopyranose main chain. The coupling constant (J_H1,2) of galactose was 3.4 Hz, indicating that galactose has an α-linkage. The main chain mannose was found to connect through the 1 → 4 linkage, because of the appearance of the correlation signals MH1-MC4, and MC1-MH4 in the HMBC spectrum due to the long-range correlation signals between two neighboring mannopyranose residues through the M4-O-M1 bond. Although the main chain mannose J_H1,2 was not observed, probably because of the high molecular weight, the specific rotation of LG with a higher proportion of mannose was low, [α]_D²⁵ = +10.8°, compared with that of FG with a lower proportion of mannose, [α]_D²⁵ = +90.5°, suggesting that the mannose in the main chain had a α-linkage. These results suggest that the galactomannans comprise a (1 → 4)-β-mannopyranosidic main chain connected with more than two (1 → 4)-α-galactopyranosidic side chains, in addition to the single galactopyranose side chain, at C6 of the mannopyranose main chain.     

The Use of Amphipols for NMR Structural Characterization of 7-TM Proteins

While amphipols have been proven useful for refolding of seven transmembrane helical (7-TM) proteins including G-protein-coupled receptors (GPCRs) and it could be shown that an amphipol environment is in principle suitable for NMR structural studies of the embedded protein, high-resolution NMR insights into amphipol refolded and isotopically labeled GPCRs are still very limited. Here we report on the recent progress toward NMR structural studies of the melanocortin-2 and -4 receptors, two class A GPCRs which so far have not been reported to be incorporated into an amphipol environment. Making use of the established 7-TM protein bacteriorhodopsin (BR) we initially tested and optimized amphipol refolding conditions. Most promising conditions were transferred to the refolding of the two melanocortin receptors. Analytical-scale refolding experiments on the melanocortin-2 receptor show very similar behavior to the results obtained on BR. Using cell-free protein expression we could generate sufficient amounts of isotopically labeled bacteriorhodopsin as well as melanocortin-2 and -4 receptors for an initial NMR analysis. Upscaling of the amphipol refolding protocol to protein amounts needed for NMR structural studies was, however, not straightforward and impeded detailed NMR insights for the two GPCRs. While well-resolved and dispersed NMR spectra could only be obtained for bacteriorhodopsin, a comparison of NMR data recorded on the melanocortin-4 receptor in SDS and in an amphipol environment indicates that amphipol refolding induces larger structural modifications in the receptor.     

Characterization of human saposins by NMR spectroscopy

Saposins are lipid-binding and membrane-perturbing glycoproteins of the mammalian lysosomes involved in sphingolipid and membrane digestion. Although the four human saposins (Saps), A-D, are sequence-related, they are responsible for the activation of different steps in the cascade of lysosomal glycosphingolipid degradation. Saposin activity is maximal under acidic conditions, and the pH dependence of lipid and membrane binding has been assigned to conformational variability. We have employed solution NMR spectroscopy to all four (15)N-labeled human saposins at both neutral and acidic pH. Using backbone NOEs and residual dipolar couplings, the "saposin fold" comprising five alpha-helices was confirmed for Sap-A, Sap-C, and Sap-D. Structural variations within these proteins are in the order of variations between the known structures of Sap-C and NK-lysin. In contrast, Sap-B yielded spectra of very poor quality, presumably due to conformational heterogeneity and molecular association. Sap-D exists in a slow dynamic equilibrium of two conformational states with yet unknown function. At pH 4.0, where all saposins are highly unstable, Sap-C undergoes a transition to a specific dimeric state, which is likely to resemble the structure recently found in both Sap-C in a detergent environment and crystals of Sap-B.     

Understanding the Mechanism of Prosegment-catalyzed Folding by Solution NMR Spectroscopy

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.     

Periodate Oxidation of Some Carbohydrates as Examined by NMR Spectroscopy

Periodate oxidation of some sugar alcohols, methyl glycosides and a synthetic glucan in an amount of 5 ~ 20 mg was performed in ca. 0.2 ~ 0.4 ml of D₂O involving NaIO₄ (1.5 ~ 2.0 moles excess) in a NMR sample tube, and the reaction products were examined in the course of oxidation by NMR spectroscopy.

In addition to proton signals of formyl and formaldehyde (in acetal), proton signals at hemiacetal carbons were identified in the periodate oxidation. Splitting and change in O-methyl and N-acetate-methyl signals indicated presence of more than one structures for each of the reaction products in the periodate oxidations of methyl α-d-glucopyranoside and methyl N-acetyl-α-d-glucosaminide. A condensation product was detected in the periodate oxidation of glycolaldehyde, d,l-glyceraldehyde and d-galactitol. A synthetic glucan was found to have a structure of 1,6-linkage in a DP = 15?17.     

Characterization of Danio rerio Mn(2+)-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase, the Structural Prototype of the ADPRibase-Mn-Like Protein Family

The ADPRibase-Mn-like protein family, that belongs to the metallo-dependent phosphatase superfamily, has different functional and structural prototypes. The functional one is the Mn(2+)-dependent ADP-ribose/CDP-alcohol diphosphatase from Rattus norvegicus, which is essentially inactive with Mg(2+) and active with low micromolar Mn(2+) in the hydrolysis of the phosphoanhydride linkages of ADP-ribose, CDP-alcohols and cyclic ADP-ribose (cADPR) in order of decreasing efficiency. The structural prototype of the family is a Danio rerio protein with a known crystallographic structure but functionally uncharacterized. To estimate the structure-function correlation with the same protein, the activities of zebrafish ADPRibase-Mn were studied. Differences between zebrafish and rat enzymes are highlighted. The former showed a complex activity dependence on Mn(2+), significant (≈25%) Mg(2+)-dependent activity, but was almost inactive on cADPR (150-fold less efficient than the rat counterpart). The low cADPR hydrolase activity agreed with the zebrafish genome lacking genes coding for proteins with significant homology with cADPR-forming enzymes. Substrate-docking to zebrafish wild-type protein, and characterization of the ADPRibase-Mn H97A mutant pointed to a role of His-97 in catalysis by orientation, and to a bidentate water bridging the dinuclear metal center as the potential nucleophile. Finally, three structural elements that delimit the active site entrance in the zebrafish protein were identified as unique to the ADPRibase-Mn-like family within the metallo-dependent phosphatase superfamily.     

Solid-Phase Synthesis and NMR Structural Studies of the Marine Antibacterial Cyclic Tetrapeptide: Cyclo[GSPE]

Twelve-membered head-to-tail cyclic tetrapeptides (CTPs) are rigid molecules found in nature and possess a diverse range of biological activities. A possible reason may be due to their ability to adopt rigid conformations in solution mimicking reverse-turns. Reverse-turns are common structural motifs which serve as molecular recognition sites in many protein-receptor interactions. In this paper, we describe the solid-phase synthesis of the antibacterial cyclic tetrapeptide cyclo[Gly-Ser-Pro-Glu] (cyclo[GSPE]), first isolated from the Ruegeria strain of marine bacteria by Mitova et al. (J Nat Prod 67:1178–1181, 2004). Our NMR experiments in H₂O:D₂O:DMSO (18:1:1) revealed that it possessed three conformations in an approximate ratio of 4:2:1 based on NMR amide peak intensities. 2D NMR studies and computer calculations revealed that the major conformer adopted a reverse-turn conformation and have ω torsion angles twisted by up to 2°, with two transoid amide bonds between Gly-Ser, Pro-Glu and two cisoid amide bonds between Ser-Pro, Glu-Gly in a cis–trans-cis–trans (ctct) pattern. This supports previous reports that majority of CTPs adopt a ctct pattern when dissolved in hydrogen-bond disrupting solvents (Che and Marshall in J Med Chem 49:111–124, 2006 and references cited therein). An ensemble of ten lowest-energy-minimised 3D structures generated using XPLOR-NIH software revealed that cyclo[GSPE] possessed a rigid backbone ring scaffold. The remaining two minor conformers were present in quantities too low for NMR structural studies.     

Characterization of insoluble calcium alginates by solid-state NMR

Three series of 9 insoluble calcium alginate powders with different average calcium contents (1.5, 3.5 and 8%, w/w) are investigated by means of ¹³C solid-state NMR spectroscopy. The effect of the increased calcium content on the determination of the mannuronate (M) to guluronate (G) ratio from spectral deconvolution of the ¹³C CP/MAS spectra is discussed, and the variations observed are commented in function of possible structural modifications related to the interaction with the divalent cations. The possibility of using solid-state NMR spectroscopy for the quantification of the calcium content in unknown alginate samples is explored performing principal component analysis (PCA) of the spectra. The results obtained show that a clear separation of alginates with slightly different calcium content is possible. The proposed method relies on the sole use of the chemical shifts of the signals corresponding to pyranose carbons, suggesting that PCA of solid-state NMR data holds promises as a rapid and undestructive method for screening the calcium content of alginate-based materials with biomedical uses.