Nils Åge Frøystein

The Membrane-bound Conformation of α-Lactalbumin Studied by NMR-monitored 1H Exchange

Abstract The interaction of bovine α-lactalbumin (BLA) with negatively charged phospholipid bilayers was studied by NMR monitored 1H exchange to characterize the conformational transition that enables a water-soluble protein to associate with and partially insert into a membrane. BLA was allowed to exchange in deuterated buffer in the absence (reference) and the presence (membrane-bound) of acidic liposomes at pH 4.5, experimental conditions that allow efficient protein–membrane interaction. After adjusting the pH to 6.0, to dissociate the protein from the membrane, reference and membrane-released samples of BLA were analysed by (F1) band-selective homonuclear decoupled total correlation spectroscopy in the αH–NH region. The overall exchange behaviour of the membrane-bound state is molten globule-like, suggesting an overall destabilization of the polypeptide. Nevertheless, the backbone amide protons of residues R10, L12, C77, K94, K98, V99 and W104 show significant protection against solvent exchange in the membrane-bound protein. We propose a mechanism for the association of BLA with negatively charged membranes that includes initial protonation of acidic side-chains at the membrane interface, and formation of an interacting site with the membrane which involves helixes A and C. In the next step these helices would slide away from each other, adopting a parallel orientation to the membrane, and would rotate to maximize the interaction between their hydrophobic residues and the lipid bilayer.

Anthocyanin trisaccharides in blue berries of Vaccinium padifolium

Delphinidin 3-O-a-rhamnoside, malvidin 3-O-(6 00 -O-a-rhamnopyranosyl-b-glucopyranoside) and the 3-O-(6 00 -O-a-rhamnopyranosyl-2 00 -O-b-xylopyranosyl-b-glucopyranosides) of cyanidin, petunidin and peonidin were isolated by various chromatographic techniques from the edible berries of Vaccinium padifolium. Their complete structures were elucidated mainly by one- and twodimensional nuclear magnetic resonance spectroscopy. Together they account for 7% of the anthocyanin content in this species. No anthocyanidin 3-triglycoside, 3-rutinoside or 3-rhamnoside had previously been found in the genus Vaccinium. The 3-O-(6 00 -O-arhamnopyranosyl-2 00 -O-b-xylopyranosyl-b-glucopyranosides) of petunidin and peonidin are novel compounds. # 2000 Elsevier

Mercuration of acetone in the acetone-mercury(II) perchlorate-2-(2′-pyridyl)quinoxaline(L) system

Abstract The 1:1 reaction of Hg(ClO 4 ) 2 ·3H 2 O with 2-(2′-pyridyl)quinoxaline(L) in acetone leads to isolation of [Hg(CH 2 COCH 3 )(ClO 4 L] 2 ; the X-ray crystal structure determination together with 1 H- and 13 C-NMR spectra reveal the presence of the coordinated 2-oxopropyl anion. L coordinates in a bidentate, highly asymmetric mode.

Interaction of manganese(II), cobalt(II) and nickel(II) with DNA oligomers studied by 1H NMR spectroscopy

Abstract The metal binding properties of three different oligodeoxyribonucleotides, one decamer and two dodecamers, have been studied by 1D and 2D 1H NMR spectroscopy. The sequential composition of the oligomers was chosen so as to provide a multitude of different binding site contexts: 5′-GACGGCCGTC (I), 5′-CGCGAATTCGCG (II), and 5′-ATGGGTACCCAT (III). Salts of three different metal ions: Mn(II), Co(II) and Ni(II), were used in titration experiments. The binding studies were carried out under duplex conditions. The influence of paramagnetic metal ions on protons adjacent to the binding site were followed by measuring line broadening and T1 relaxation times. We have previously proposed a rule for sequence-selective metal binding to G-N7 in oligodeoxyribonucleotides where the metal prefer the 5′-guanine residue in the order: 5′-GG ≥ GA > GT ≫ GC. This binding pattern is also observed in the present study for Mn(II) and Co(II). However, in sequence I Ni(II) does not follow the established selectivity rule showing greater affinity for 5′-G in the context 5′-GC than in the context 5′-GA. For II and III Ni(II) ions are seen to follow the selectivity rule. Fluctuation in the nucleophilicity of G-N7 along the duplex is probably a consequence of sequence-dependent variation in π-stacking interaction between base residues.

Human Protein N-terminal Acetyltransferase hNaa50p (hNAT5/hSAN) Follows Ordered Sequential Catalytic Mechanism COMBINED KINETIC AND NMR STUDY

Background: Nα-Acetylation is catalyzed by N-terminal acetyltransferases (NATs). The reaction mechanisms of NATs are unknown. hNaa50p is a member of the human NAT family. Results: Kinetic parameters and product inhibition patterns were determined. Acetyl-CoA binding induced conformational changes facilitating peptide binding. Conclusion: hNaa50p most likely utilizes the Theorell-Chance mechanism. Significance: Bisubstrate inhibitors, mimicking a ternary complex, should function as specific inhibitors of human NATs. Nα-Acetylation is a common protein modification catalyzed by different N-terminal acetyltransferases (NATs). Their essential role in the biogenesis and degradation of proteins is becoming increasingly evident. The NAT hNaa50p preferentially modifies peptides starting with methionine followed by a hydrophobic amino acid. hNaa50p also possesses Nϵ-autoacetylation activity. So far, no eukaryotic NAT has been mechanistically investigated. In this study, we used NMR spectroscopy, bisubstrate kinetic assays, and product inhibition experiments to demonstrate that hNaa50p utilizes an ordered Bi Bi reaction of the Theorell-Chance type. The NMR results, both the substrate binding study and the dynamic data, further indicate that the binding of acetyl-CoA induces a conformational change that is required for the peptide to bind to the active site. In support of an ordered Bi Bi reaction mechanism, addition of peptide in the absence of acetyl-CoA did not alter the structure of the protein. This model is further strengthened by the NMR results using a catalytically inactive hNaa50p mutant.

Myricetin 3-rhamnosyl(1→6)galactoside from Nymphaéa x marliacea

Abstract The novel flavonol, myricetin 3- O - α -rhamnopyranosyl(1→6) β -galactopyranoside was isolated from leaves of the water lily Nymphaea x marliacea (white petals), while four flavonols, quercetin and the 3′-xylosides of myricetin, quercetin and quercetin 3-methyl ether were isolated from red petals of the variety Escarboucle . Their structures were elucidated by homo- and heteronuclear two-dimensional NMR techniques, other spectroscopic techniques and chromatography. This is the first report of flavonol 3′-xylosides as flower pigments.

Mutation of the highly conserved Ser-40 of the HIV-1 p6 gag protein to Phe causes the formation of a hydrophobic patch, enhances membrane association, and polyubiquitination of Gag.

The HIV-1 p6 Gag protein contains two late assembly (l-) domains that recruit proteins of the endosomal sorting complex required for transport (ESCRT) pathway to mediate membrane fission between the nascent virion and the cell membrane. It was recently demonstrated that mutation of the highly conserved Ser-40 to Phe (S40F) disturbs CA-SP1 processing, virus morphogenesis, and infectivity. It also causes the formation of filopodia-like structures, while virus release remains unaffected. Here, we show that the mutation S40F, but not the conservative mutation to Asp (S40D) or Asn (S40N), augments membrane association, K48-linked polyubiquitination, entry into the 26S proteasome, and, consequently, enhances MHC-I antigen presentation of Gag derived epitopes. Nuclear magnetic resonance (NMR) structure analyses revealed that the newly introduced Phe-40, together with Tyr-36, causes the formation of a hydrophobic patch at the C-terminal α-helix of p6, providing a molecular rationale for the enhanced membrane association of Gag observed in vitro and in HIV-1 expressing cells. The extended exposure of the S40F mutant to unidentified membrane-resident ubiquitin E3-ligases might trigger the polyubiquitination of Gag. The cumulative data support a previous model of a so far undefined property of p6, which, in addition to MA, acts as membrane targeting domain of Gag.

Binding of copper(II) and manganese(II) to 9-methylpurine studied by nuclear magnetic resonance and potentiometric titration

Abstract The binding of Cu(II) and Mn(II) to 9-methylpurine has been examined by proton NMR and potentiometric titration. The spin-lattice relaxation rates of ring protons H2, H6 and H8, used as input to the Solomon-Bloembergen equation, give a binding pattern which qualitatively agrees with crystallographic data. At neutral pH Cu(II) and Mn(II) binding occurs at both N1 and N7 with a preference for N7 (66%). At acidic pH, where N1 is the predominant protonation site, a further shift towards N7 binding is observed. These results are not in complete agreement with those based on comparison of N1/N7 basicity.

Thermal stability of chicken brain α-spectrin repeat 17: a spectroscopic study

Spectrin is a rod-like multi-modular protein that is mainly composed of triple-helical repeats. These repeats show very similar 3D-structures but variable conformational and thermodynamical stabilities, which may be of great importance for the flexibility and dynamic behaviour of spectrin in the cell. For instance, repeat 17 (R17) of the chicken brain spectrin α-chain is four times less stable than neighbouring repeat 16 (R16) in terms of ∆G. The structure of spectrin repeats has mainly been investigated by X-ray crystallography, but the structures of a few repeats, e.g. R16, have also been determined by NMR spectroscopy. Here, we undertook a detailed characterization of the neighbouring R17 by NMR spectroscopy. We assigned most backbone resonances and observed NOE restraints, relaxation values and coupling constants that all indicated that the fold of R17 is highly similar to that of R16, in agreement with previous X-ray analysis of a tandem repeat of the two domains. However, 15N heteronuclear NMR spectra measured at different temperatures revealed particular features of the R17 domain that might contribute to its lower stability. Conformational exchange appeared to alter the linker connecting R17 to R16 as well as the BC-loop in close proximity. In addition, heat-induced splitting was observed for backbone resonances of a few spatially related residues including V99 of helix C, which in R16 is replaced by the larger hydrophobic tryptophan residue that is relatively conserved among other spectrin repeats. These data support the view that the substitution of tryptophan by valine at this position may contribute to the lower stability of R17.

Ditetradecyl selenide, (C14H29)2Se, a new chemical shift reference compound for 77Se NMR

Ditetradecyl selenide, (C14H29)2Se, is suggested as an alternative to Me2Se as a reference compound for 77Se NMR in organic solvents. This selenide, a crystalline, non‐odorous and stable compound, is readily prepared and purified and is sufficiently soluble in most of the usual organic solvents at room temperature to give a satisfactory 77Se signal; in methanol, acetonitrile and dimethyl sulfoxide temperatures above ∼310 K are required. The chemical shifts, +162.3 ppm in CDCl3 at 298 K relative to a 60% solution of Me2Se in CDCl3, defined as 0.0 ppm, and +167.2 ppm relative to a 0.25 M solution of Me2Se in CDCl3, is independent of the concentration in the 0.01–0.40 M range. The shift in CDCl3 increases fairly linearly with temperature in the 270–345 K range with a slope of ∼0.075 ppm K−1. The shift appears to be less dependent upon the solvent than the shift of Me2Se. Copyright