Niels H. Andersen

Crop manuring and intensive land management by Europe’s first farmers

The spread of farming from western Asia to Europe had profound long-term social and ecological impacts, but identification of the specific nature of Neolithic land management practices and the dietary contribution of early crops has been problematic. Here, we present previously undescribed stable isotope determinations of charred cereals and pulses from 13 Neolithic sites across Europe (dating ca. 5900–2400 cal B.C.), which show that early farmers used livestock manure and water management to enhance crop yields. Intensive manuring inextricably linked plant cultivation and animal herding and contributed to the remarkable resilience of these combined practices across diverse climatic zones. Critically, our findings suggest that commonly applied paleodietary interpretations of human and herbivore δ15N values have systematically underestimated the contribution of crop-derived protein to early farmer diets.

A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein.

Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations. Even though some unfolded and partially folded proteins are now known to possess biological function or to be involved in aggregation phenomena associated with disease states, experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a ‘Trp-cage’ miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR pulse-labelling experiments that involve rapid in situ protein refolding. These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.

The Trp-cage: optimizing the stability of a globular miniprotein

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).

The identification of sesquiterpene hydrocarbons from gas-liquid chromatography retention data

Abstract The identification of sesquiterpenes by gas-liquid chromatography is evaluated. Retention indices (obtained using sesquiterpene standards rather than n-alkanes) were found to be the most reproducible form of GLC data. Retention data for fifty-five sesquiterpenes and a number of saturated hydrocarbons obtained by hydrogenation of sesquiterpenes are reported.

Hevein: NMR assignment and assessment of solution-state folding for the agglutinin-toxin motif.

The first high-resolution solution-state structure of a member of the toxin-agglutinin folding motif with the WGA disulfide linkage is presented. The 1H NMR spectrum of hevein has been 100% assigned from residue 2 through residue 43, the C-terminus, using two-dimensional correlation and NOE spectroscopy. During the course of the NOESY analysis, the three-dimensional structural features of hevein were derived, using nonstereospecific distance constraints (with tight bounds) for XPLOR simulated annealing followed by unconstrained relaxation in the CHARMm force field, at two levels of long-range constraint density. In addition, a large number of low-bound-only constraints, corresponding to unobserved NOEs, were used in both refinements. The first structure elucidation employed a total of 180 distance constraints (60 of which were medium or long range, i/i+n with n < or = 2). The second refinement employed 244 (101 medium or long range) constraints: some conformation-insensitive intraresidue constraints were deleted, two misassigned long-range constraints were corrected, and 41 new i/i+n (n > or = 2) constraints were added. The average bounds precisions of the two refinements were comparable (+/- 0.44 A) and significantly tighter than those that result when a universal low bound corresponding to the sum of the van der Waals radii was used. (The more conservative treatment of NOEs gave the same final structure but required a higher constraint density before assignment errors would stand out during the refinement.) Constraint density also has a significant influence on convergence and accuracy using tight constraints. The study demonstrates that convergence within an ensemble of solution structures is not a dependable criterion for either the accuracy or precision of the derived structure. The best fitting conformers from the refinement at the higher constraint density bear a greater similarity to the solid-state structure of the domains of wheat germ agglutinin (0.95 A rmsd over residues 2-32) than to the recently reported 2.8-A X-ray structure of hevein (1.25 A rmsd over residues 2-32, 2.83 A rmsd over residues 2-42). The consensus conformer from the solution data is defined to a backbone rmsd of < 0.6 A over the full sequence for which NMR data could be collected.(ABSTRACT TRUNCATED AT 400 WORDS)

Hairpin folding rates reflect mutations within and remote from the turn region

Hairpins play a central role in numerous protein folding and misfolding scenarios. Prior studies of hairpin folding, many conducted with analogs of a sequence from the B1 domain of protein G, suggest that faster folding can be achieved only by optimizing the turn propensity of the reversing loop. Based on studies using dynamic NMR, the native GB1 sequence is a slow folding hairpin \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}(k_{{\mathrm{F}}}^{278}=1.5{\times}10^{4}/{\mathrm{s}})\end{equation*}\end{document}. GB1 hairpin analogs spanning a wide range of thermodynamic stabilities \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}({\Delta}G_{{\mathrm{U}}}^{298}=-3.09{\;}{\mathrm{to}}+3.25{\;}{\mathrm{kJ}}/{\mathrm{mol}})\end{equation*}\end{document} were examined. Fold-stabilizing changes in the reversing loop can act either by accelerating folding or retarding unfolding; we present examples of both types. The introduction of an attractive side-chain/side-chain Coulombic interaction at the chain termini further stabilizes this hairpin. The 1.9-fold increase in folding rate constant observed for this change at the chain termini implies that this Coulombic interaction contributes before or at the transition state. This observation is difficult to rationalize by “zipper” folding pathways that require native turn formation as the sole nucleating event; it also suggests that Coulombic interactions should be considered in the design of systems intended to probe the protein folding speed limit.

Sesquiterpene constituents of two liverworts of genus diplophyllum: Novel eudesmanolides and cytotoxicity studies for enantiomeric methylene lactones

Abstract The steam distillates (or hexane extracts) of the liverworts Diplophyllum albicans (L.) Dum. and D. taxifolium (Wahl.) Dum. are largely a mixture of sesquiterpenes. Both species elaborate, among the hydrocarbons, mainly ent-α-selinene (8a) and ent-selina-4,11-diene (8b) together with anastreptene (1) and β-elemene (9). The major component of each essential oil was diplophyllin (10), a novel ent-eudesmanolide. The structure of diplophyllin was established by correlation with tetrahydro-isoalantolactone (17). In addition, the enantiomers of diplophyllin and diplophyllolide-A (16) were obtained on acid isomerization of isoalantolacetone (14a). Diplophyllin shows significantly greater activity against human epidermoid carcinoma (KB cell culture, ED50⋍ 8 μ g ml ) than its enantiomer: the first demonstration (to our knowledge) of optical selectivity for this type of cytotoxicity. Among the more polar constituents of D. albicans were 9α-acetoxy-diplophyllin, and several sesquiterpene alcohols including albicanol (22).

Efforts toward deriving the CD spectrum of a 310 helix in aqueous medium

There have been two recent reports suggesting that 310 helices can be distinguished from α helices by circular dichroism. The differentiating feature is stated to be a [θ]222:[θ]208 ratio (R2) distinctly smaller than unity. This has been reported for a Cα,α′‐disubstituted homooctamer [Toniolo et al. (1996), J. Am. Chem. Soc. 118, 2744–27451 and for alanine‐rich systems of 16–21 residue length with modest fractional helicity [Millhauser (1995) Biochemistry 34, 3873–38771. We report here the changes in the CD spectrum produced by inserting aminoisobutyric acid (Aib) residues into the helical domain of human pancreatic amylin. In order to examine this effect at comparable net fractional helicities, CD spectra were measured for each species during the course of a helicity titration by trifluoroethanol addition. The addition of five Aib residues gave results of particular interest. At low net fractional helicity, this Aib‐rich system displays a diminished (circa 208 nm) rotational strength versus the less Aib‐rich species. However, NMR data and comparisons of CD difference spectra suggest that fluoroalcohol‐induced extension of the short Aib‐rich helix is in the form of an α helix. Given the diminished intensity of the minimum at 208 nm at low net helicity when 310 conformations should contribute, we urge extreme caution in using a [θ]222:[θ]208 ratio smaller than unity as a diagnostic for 310 helices.

Stabilizing capping motif for β-hairpins and sheets

Although much has been learned about the design of models of β-sheets during the last decade, modest fold stabilities in water and terminal fraying remain a feature of most β-hairpin peptides. In the case of hairpin capping, nature did not provide guidance for solving the problem. Some observations from prior turn capping designs, with further optimization, have provided a generally applicable, “unnatural” beta cap motif (alkanoyl-Trp at the N terminus and Trp-Thr-Gly at the C terminus) that provides a net contribution of 6 + kJ/mol to β-hairpin stability, surpassing all other interactions that stabilize β-hairpins including the covalent disulfide bond. The motif, made up entirely of natural residues, is specific to the termini of antiparallel β-strands and reduces fraying at the ends of hairpins and other β-sheet models. Utilizing this motif, 10- to 22-residue peptide scaffolds of defined stereochemistry that are greater than 98% folded in water have been prepared. The β-cap can also be used to staple together short antiparallel β-strands connected by a long flexible loop.

Terpenes and sesquiterpenes of Chamaecyparis nootkatensis leaf oil.

Abstract The composition of the leaf oil of Chamaecyparis nootkatensis is reported. The terpene fraction consists of (−)-α-pinene, (+)-3-carene and (+)-limonene together with small amounts of (−)-β-pinene, myrcene and two unidentified hydrocarbons. The sesquiterpene fraction is largely a mixture of levoratory α-, β-, and γ-curcumenes with lesser amounts of (−)-α-copaene, (+)-α-ylangene, (+)-longifolene, (+)-δ-cadinene, (+)-γ-cadinene, (+)-β-bisabolene, β-farnesene, (+)-trans-nerolidol and three substances which have not been fully characterized. One of these appears to be a mixture of diastereomeric “calamenenes”; the others are new sesquiterpenes which we designate as α- and β-alaskene. Valencene and nootkatene, previously reported from the heartwood oil of this species, were not detected in the leaf oil. The biogenetic and chemotaxonomic implications of these results are discussed.