Spectral Changes of the γ-Irradiated Glucose Solution

β-1,3-Xylanase was purified to gel electrophoretic homogeneity and 83-fold from a cell-free culture fluid of Vibrio sp. XY-214 by ammonium sulfate precipitation and successive chromatographies. The enzyme had a pl of 3.6 and a molecular mass of 52 kDa. The enzyme had the highest level of activity at pH 7.0 and 37°C. The enzyme activity was completely inhibited by Cu²⁺, Hg²⁺, and N-bromosuccinimide. The enzyme hydrolyzed β-1,3-xylan to produce mainly xylotriose and xylobiose but did not act on xylobiose, p-nitrophenyl-β-D-xyloside, β-1,4-xylan, β-1,3-glucan, or carboxymethyl cellulose.     

Polymerization of β-amino Acids in Aqueous Solution

We have compared carbonyl diimidazole (CDI) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as activating agents for the oligomerization of negatively-charged - and -amino acids in homogeneous aqueous solution. -Amino acids can be oligomerized efficiently using CDI, but not by EDAC. -Amino acids can be oligomerized efficiently using EDAC, but not by CDI. Aspartic acid, an - and -dicarboxylic acid is oligomerized efficiently by both reagents. These results are explained in terms of the mechanisms of the reactions, and their relevance to prebiotic chemistry is discussed.     

Solution NMR studies of Aβ monomer dynamics

Aβ is widely recognized as a key molecule in Alzheimer's disease, causing neurotoxicity through Aβ aggregates such as Aβ oligomers and fibrils. Aβ40 and Aβ42, composed of 40 and 42 residues, respectively, are the major Aβ species in human brain. Aβ42 aggregates much faster than Aβ40 but the mechanism of such difference in aggregation propensity is poorly understood. Using NMR spin relaxation, we have shown that Aβ40 and Aβ42 monomers have different dynamics in both backbone and sidechain on the ps-ns time scale. Aβ42 is more rigid in C-terminus in both backbone and sidechain while Aβ40 has more rigid methyl groups in the central hydrophobic cluster (CHC: Aβ17-21). These observations are consistent with differences in the major conformations of Aβ40 and Aβ42 monomers derived from replica exchange MD (REMD). To further demonstrate the relevance of dynamics in aggregation mechanism, a perturbation was introduced to Aβ42 in the form of M35 oxidation. After M35 side chain oxidation to sulfoxide, Aβ42 experiences Aβ40-like changes in dynamics. At the same time, M35 oxidation causes dramatic reduction in Aβ42 aggregation rate. These data have thus established an important role for protein dynamics in the mechanism of Aβ aggregation.     

Solution NMR studies of polytopic α-helical membrane proteins

NMR spectroscopy has established itself as one of the main techniques for the structural study of integral membrane proteins. Remarkably, over the last few years, substantial progress has been achieved in the structure determination of increasingly complex polytopical α-helical membrane proteins, with their size approaching ～100kDa. Such advances are the result of significant improvements in NMR methodology, sample preparation and powerful selective isotope labelling schemes. We review the requirements facilitating such work based on the more recent solution NMR studies of α-helical proteins. While the majority of such studies still use detergent-solubilized proteins, alternative more native-like lipid-based media are emerging. Recent interaction, dynamics and conformational studies are discussed that cast a promising light on the future role of NMR in this important and exciting area.     

Solution dynamics of the 1,2,3,4,6-penta-O-acetyl-α-D-idopyranose ring

The anticoagulant properties of heparin are thought to derive from the inhibition of thrombin and other coagulation-related proteases by the binding of heparin to cofactors such as antithrombin III and heparin cofactor II. The apparent minimum native heparin sequence which can bind to antithrombin III is a highly sulfated pentasaccharide which contains a 2-O-sulfo-α-L-idopyranosyluronic acid residue. The idopyranosyl residue has the unusual property of existing in the solution state as a mixture of ring conformers. Whereas most hexopyranose sugars exist as a single chair conformer (eg D-glucose exists overwhelmingly as a 4C1 chair), the idopyranosyl ring is known to rapidly exchange between at least two and often more distinct conformations, depending on type and number of substituents (hydroxyl, carboxyl, sulfate, etc.) and solvent conditions (solvent pH, salt concentration, temperature). It is believed that this flexibility of the idopyranosyl residue in heparin is related to its binding specificity. In the past, coupling constants and molecular dynamics have been used to estimate the relative populations of conformers in iduronate and related compounds. Here we report extensive NMR measurements, including line shape analysis, T1ρ measurements, T1 and NOE measurements and spectral density mapping, which have been used to study the dynamics of conformer interconversion in model compounds related to idose and glucose. The findings presented here indicate that 1,2,3,4,6-peneta-O-acetyl-α-D-idopyranose can be reasonably well described as existing in a two-state equilibrium consisting of the 4C1 and 0S2 conformers. 13C NMR line shape analysis yields a ΔH+ of 40 kJ mol-1 and a ΔS‡ of 31 J mol-1 K-1 for the 4C1→0S2 interconversion and a ΔH‡ of 31 kJ mol-1 and a ΔS‡ of 13 J mol-1K-1 for the 0S2→4C1 interconversion. This corresponds to exchange rates of 22 and 128 MHz, respectively, at room temperature. This revised version was published online in November 2006 with corrections to the Cover Date.     

Solution conditions define morphological homogeneity of α-synuclein fibrils

The intrinsically disordered human α-synuclein (αSyn) protein exhibits considerable heterogeneity in in vitro fibrillization reactions. Using atomic force microscopy (AFM) we show that depending on the solvent conditions, A140C mutant and wild-type αSyn can be directed to reproducibly form homogeneous populations of fibrils exhibiting regular periodicity. Results from Thioflavin-T fluorescence assays, determination of residual monomer concentrations and native polyacrylamide gel electrophoresis reveal that solvent conditions including EDTA facilitate incorporation of a larger fraction of monomers into fibrils. The fibrils formed in 10 mM Tris–HCl, 10 mM NaCl and 0.1 mM EDTA at pH 7.4 display a narrow distribution of periodicities with an average value of 102 ± 6 nm for the A140C mutant and 107 ± 9 nm for wt αSyn. The ability to produce a homogeneous fibril population can be instrumental in understanding the detailed structural features of fibrils and the fibril assembly process. Moreover, the availability of morphologically well-defined fibrils will enhance the potential for use of amyloids as biological nanomaterials.     

Radiation-induced Ring Cleavage of β-Cyclodextrin in Oxygen-free Aqueous Solution

The degraded products of β-cyclodextrin (β-CD) by γ-irradiation in aqueous solution were identified as six oligosaccharides, such as glucose, maltose, maltotriose, maltotetraose, malto-pentaose and maltohexaose, by the gel-filtration method on Biogel P-2 at 60°C. Analysis of the hyperfine structure of the ESR spectrum observed in the oxidation of β-CD with Ti³⁺-hydrogen peroxide system indicated that a radical was formed mainly by hydrogen abstraction at C-5 of a glucose residue. A mechanism of the ring cleavage of β-CD by γ-irradiation in oxygen-free aqueous solution was proposed.     

Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX

The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane β-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine. Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used, some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the dynamics of the barrel.     

Solution Structure and Acid-Base Properties of Reduced α-Conotoxin MI

The reduced derivative of α-conotoxin MI, a 14 amino acid peptide is characterized by NMR-pH titrations and molecular dynamics simulations to determine the protonation constants of the nine basic moieties, including four cysteine thiolates, and the charge-dependent structural properties. The peptide conformation at various protonation states was determined. The results show that the disulfide motifs in the native globular α-conotoxin MI occur between those cysteine moieties that exhibit the most similar thiolate basicities. Since the basicity of thiolates correlates to its redox potential, this phenomenon can be explained by the higher reactivity of the two thiolates with higher basicities. The folding of the oxidized peptide is further facilitated by the loop-like structure of the reduced form, which brings the thiolate groups into sufficient proximity. The 9 group-specific protonation constants and the related, charge-dependent, species-specific peptide structures are presented.     

Solution structure of β2-microglobulin and insights into fibrillogenesis

The solution structure of human β₂-microglobulin (β₂-m) was determined by ¹H NMR spectroscopy and restrained modeling calculations. Compared to the crystal structure of type I major histocompatibility complex (MHC-I), where the protein is associated to the heavy-chain component, several differences are observed, i.e., increased separation between strands A and B, displacements of strand C′ and loop DE, shortening of strands D and E. These modifications can be considered as the prodromes of the amyloid transition. Even minor charge changes in response to pH, as is the case with H31 imidazole protonation, trigger the transition that starts with unpairing of strand A. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu²⁺ binding which is shown to occur primarily at H31. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches via surface charge cluster. Mutants or truncated forms of β₂-m can be designed to remove the instability from H31 titration or to enhance the instability through surface charge suppression. By monitoring the conformational evolution of wild-type protein and variants thereof, either in response or absence of external perturbation, valuable insights into intermediate structure and fibrillogenesis mechanisms are gained.     

Solution properties of Cu(II)-L-α-alaninehydroxamic acid

In the aqueous solution of copper(II) ions, bidentate L-α-alaninehydroxamic acid (CH₃CH(NH₂)-CONHOHHL) binds cupric ion forming of monodimeric and bis(L-α-alaninehydroxamato)copper(II) complexes. These complexes were studied by potentiometric, ESR and spectrophotometric methods.The ESR studies provide important evidence for the formation of different Cu(II) complexes with L-α-alaninehydroxamic acid, depending on pH. The ESR spectra can be used to follow the appearance of the individual complexes, to estimate the coordination sphere around Cu(II) and to observe the equilibria between different complexes.The solution electronic spectra are reported. The experimental curve was resolved into precise- positioned absorption bands by Gaussian analysis for the bis(L-α-alaninehydroxamato)copper(II) species. These data were used in a weak tetragonal ligand field model to calculate ligand field parameters.The distribution and the relevant stability constants of species present in aqueous solutions were obtained by analytical potentiometry.     

The role of corridors in conservation: Solution or bandwagon?

Corridors are currently a major buzzword in conservation biology and landscape ecology. These linear landscape features may perform numerous functions, but it is their role in facilitating movement of fauna that has attracted much recent debate. The database supporting the idea of corridors acting as faunal conduits is remarkably small, and few studies have actually demonstrated that movement along corridors is important for any given species. Such data are very difficult to obtain, and conservation biologists are thus faced with the problem of whether to recommend the allocation of resources to corridors on the assumption that they may be important.     

The Solution NMR Structure of 2′-O-Methyl CGCGCG RNA Duplex

Abstract

Complete assignments of nonexchangeable protons in ¹H NMR spectra of 2′-O-methyl-CGCGCG complemented by its analysis of ¹³C and ³¹P NMR spectra revealed A-RNA double helical structure in low salt solution.     

Guest-Host Chemistry with Dendrimers—Binding of Carboxylates in Aqueous Solution

Recognition and binding of anions in water is difficult due to the ability of water molecules to form strong hydrogen bonds and to solvate the anions. The complexation of two different carboxylates with 1-(4-carbomethoxypyrrolidone)-terminated PAMAM dendrimers was studied in aqueous solution using NMR and ITC binding models. Sodium 2-naphthoate and sodium 3-hydroxy-2-naphthoate were chosen as carboxylate model compounds, since they carry structural similarities to many non-steroidal anti-inflammatory drugs and they possess only a limited number of functional groups, making them ideal to study the carboxylate-dendrimer interaction selectively. The binding stoichiometry for 3-hydroxy-2-naphthoate was found to be two strongly bound guest molecules per dendrimer and an additional 40 molecules with weak binding affinity. The NOESY NMR showed a clear binding correlation of sodium 3-hydroxy-2-naphthoate with the lyophilic dendrimer core, possibly with the two high affinity guest molecules. In comparison, sodium 2-naphthoate showed a weaker binding strength and had a stoichiometry of two guests per dendrimer with no additional weakly bound guests. This stronger dendrimer interaction with sodium 3-hydroxy-2-naphthoate is possibly a result of the additional interactions of the dendrimer with the extra hydroxyl group and an internal stabilization of the negative charge due to the hydroxyl group. These findings illustrate the potential of the G4 1-(4-carbomethoxy) pyrrolidone dendrimer to complex carboxylate guests in water and act as a possible carrier of such molecules.     

NMR Investigations of the Solution Conformations of Deoxynucleotidyl (3′-5′) Arabinonucleosides

Abstract

The solution conformations of all eight deoxynucleotidyl (3′-5′) arabinonucleosides containing 9-B-D-arabinofuranosyladenine and 1-B-D-arabinonfuranosylcytosine have been analyzed by NMR methods and compared to dinucleoside monophosphates containing the corresponding deoxyriboside units.     

Structure of the Nidogen Binding LE Module of the Laminin γ1 Chain in Solution

The structure of the single LE module between residues 791 and 848 of the laminin γ1 chain, which contains the high affinity binding site for nidogen, has been probed using NMR methods. The module folds into an autonomous domain which has a stable and unique three-dimensional (3D) structure in solution. The 3D structure was determined on the basis of 362 interproton distance constraints derived from nuclear Overhauser enhancement measurements and 39 π angles, supplemented by 5 ψ and 22 χ₁angles. The main features of the NMR structures are two-stranded antiparallel β-sheets which are separated by loops and cross-connected by four disulfide bridges. The N-terminal segment which contains the first three disulfide bridges is similar to epidermal growth factor. The C-terminal segment has an S-like backbone profile with a crossover at the last disulfide bridge and comprises two three-residue long β-strands that form an antiparallel β-sheet. The LE module possesses an exposed nidogen binding loop that projects away from the main body of the protein. The side-chains of three amino acids which are crucial for binding (Asp, Asn, Val) are all exposed at the domain surface. An inactivating Asn-Ser mutation in this region showed the same 3D structure indicating that these three residues, and possibly an additional Tyr in an adjacent loop, provide direct contacts in the interaction with nidogen.     

The Solution Structure of the C-Terminal Ig-like Domain of the Bacteriophage λ Tail Tube Protein

Immunoglobulin (Ig)-like domains are found frequently on the surface of tailed double-stranded DNA bacteriophages, yet their functional role remains obscure. Here, we have investigated the structure and function of the C-terminal Ig-like domain of gpV (gpV_C), the tail tube protein of phage λ. This domain has been predicted through sequence similarity to be a member of the bacterial Ig-like domain 2 (Big_2) family, which is composed of more than 1300 phage and bacterial sequences. Using trypsin proteolysis, we have delineated the boundaries of gpV_C and have shown that its removal reduces the biological activity of gpV by 100-fold; thus providing a definitive demonstration of a functional role for this domain. Determination of the solution structure of gpV_C by NMR spectroscopy showed that it adopts a canonical Ig-like fold of the I-set class. This represents the first structure of a phage-encoded Ig-like domain and only the second structure of a Big_2 domain. Structural and sequence comparisons indicate that the gpV_C structure is more representative of both the phage-encoded Big_2 domains and Big_2 domains in general than the other available Big_2 structure. Bioinformatics analyses have identified two conserved clusters of residues on the surface of gpV_C that may be important in mediating the function of this domain.     

Novel Modification of 5-Formyluracil by Cysteine Derivatives in Aqueous Solution‡

Abstract

Reactivities of 5-formyl-2′-deoxyuridine (fdU) and its 5′-monophosphate (fdUMP) to amino acids, amines and thiol compounds in neutral aqueous solution have been studied to elucidate the postmodification of the 5-formyluracil (fU) moiety in cells. fdU and fdUMP specifically reacted with cysteine and its analogs to form thiazolidine derivatives. The reaction involved condensation of the formyl group of fU with both α-NH₂ (or NH₂ at the equevalent position) and SH groups of cysteine derivatives.

     

Preparation of an Octadeoxyribonucleoside Heptaphosphorothioate by the Phosphotriester Approach in Solution†

Abstract

The synthesis of the octadeoxyribonucleoside heptaphosphorothioate, d[Tp(s)Tp(s)Gp(s)Gp(s)Gp(s)Gp(s)Tp(s)T] by the phosphotriester approach in solution is described. The phosphorothioate internucleotide linkages are protected by the S-(2-cyanoethyl) group and 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-1H-triazole (MSNT) is used as the coupling agent. A block synthesis strategy (2 + 2 → 4 and 4 + 4 → 8) is followed.