HSQC

The chemical structure of the milled-wood lignin isolated from Paulownia fortunei wood was investigated. The lignins were characterized by analytical pyrolysis and two-dimensional NMR that indicated a predominance of guaiacyl (G) over syringyl (S) units, and only showed small amounts of p-hydroxyphenyl (H) units, with a H:G:S molar ratio of 1:59:40. The heteronuclear single quantum correlation (HSQC) NMR spectrum gave additional information about the relative abundances of the different inter-unit linkages present in the lignin structure. Paulownia lignin showed a predominance of -O-4� aryl ether linkages (62% of total side-chains), followed by � resinol-type (12%) and -5 � phenylcoumaran-type structures (11%) and with lower amounts of other condensed structures such as spirodienone (3%) and dibenzodioxocin (2%) structures. The high lignin content (ca. 23% Klason lignin), together with the low S/G ratio and the abundance of condensed (carbon–carbon linked) structures (together with ...

The aim of this thesis was to investigate potential ways to utilize water extracts from spruce bark. Spruce bark has been found to contain three main stilbene glucosides (astringin, isorhapontin, polydatin), which have potential in the treatment of skin aging and cosmetics applications. Since stilbene glucosides are water soluble, they are dissolved into the debarking process waters of the pulp and paper industry. These waters are not fully utilized at the moment, and being considered as a waste stream, which adds to the costs of the mill by increasing COD levels of the waste water. This waste stream could potentially be transformed to a source of stilbene glucosides, to provide additional revenue to the mill. In the experimental part, temperature effect on hot water extraction yields of industrial spruce bark was studied. Mechanical pressing of heated spruce bark was performed with a laboratory scale mechanical press to study the effect on the COD levels. Finally, bark press waters obtained from the mill were characterized, with partial purification of stilbene glucosides by ultrafiltration with 2 and 5 kDa filters. The yields were calculated by gravimetric and spectroscopic analysis. Two dimensional (2D) solution-state 1H–13C correlation NMR spectroscopy provided the structural verification of the stilbene glucosides and other compounds present in the samples. Extraction yield of industrial spruce bark was increasing 1.4 times every 20 ◦C, on average, reaching yield of 5.0% at 80 ◦C. The mechanical pressing was able to press out 26±5% of the total mass of the sample. COD level was 75,100 mg/L for 80 ◦C sample. This was two times higher compared to bark press sample of taken from a paper mill, which had COD level of 37,500 mg/L. Ultrafiltration could remove 90% of polyphenols and permeated 14% of the stilbenes of the feed. With 2D-HSQC NMR, all three major stilbene glucosides were identified from the filtrate and the feed.

Bovine IF1 is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F1F0 ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF1 (44–84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.

An NMR-based structure is presented for a 20 mer hairpin model of the SL3 stem-loop from the HIV-1 packaging signal. The stem has an A-family structure. However, the GGAG tetraloop appears to be flexible with the second (G10) and fourth (G12) bases extruded from the normal stacking arrangement. The A-base (A11) occupies a cavity large enough for it to jump rapidly between stacking upon G9 (in the loop) and G13 (from the base-pair adjacent to the loop). The H-bonding loci of G10, A11, and G12 are unoccupied in the free RNA structure. The loop should be easily adaptable to binding by the HIV-1 nucleocapsid protein or loop receptors.

The aggregation of the intrinsically disordered protein α-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of α-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to α-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric α-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of α-synuclein. The NbSyn2:α-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of α-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of α-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of α-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo.

A series of novel pyrazole integrated benzophenones (9a-j) have been designed, synthesized from 1-methyl-5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole 6. The structures of the regioisomers 6 and 7 were determined by 2D (1)H-(1)H COSY, (1)H-(13)C HSQC and (1)H-(13)C HMBC experiments. The newly synthesized compounds (9a-j) were evaluated for in vivo anti-inflammatory activity by carrageenan paw edema in rats and in vitro COX-1/COX-2 inhibition and antioxidant potential. Among the synthesized compounds, compounds 9b, 9d and 9f, were found to be active anti-inflammatory agents in addition to having potent antioxidant activity.

The physiological transient complex between cytochrome f (Cf) and cytochrome c6 (Cc6) from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by NMR spectroscopy. The binding constant at low ionic strength is 8±2mM−1, and the binding site of Cc6 for Cf is localized around its exposed haem edge. On the basis of the experimental data, the resulting docking simulations