Alex F. Drake

Reassessment of the electronic circular dichroism criteria for random coil conformations of poly(l-lysine) and the implications for protein folding and denaturation studies

The circular dichroism (CD) spectra of poly(L-lysine) in water and ethanediol/water (2:1) solutions in the temperature range -110 to 85 degrees C are presented. The results combined with vibrational CD data are interpreted in terms of a two-state conformational equilibrium with a left-handed trans polyproline II conformation being preferred at low temperatures. The relevance of these studies to the CD criteria for random-coil conformations, the study of helix-coil transitions and protein/peptide folding is pointed out.

The effect of variation of substitution on the solution conformation of heparin: a spectroscopic and molecular modelling study

The effect of variations in substitution on the conformation of iduronate-containing sequences in heparin and heparan sulphate has been studied using a series of chemically-modified heparins in which substitution with O- and N-sulphate and N-acetyl substituents has been systematically altered. Monosaccharides corresponding to residues in these modified heparins have also been investigated. The conformations of the glycosidic linkages in O- and N-desulphated re-N-acetylated heparin, O-desulphated re-N-sulphated heparin, and 6-O-desulphated re-N-sulphated heparin have been compared with those of N-desulphated re-N-acetylated heparin and of heparin itself, which have been compared with those of N-desulphated re-N-acetylated heparin and of heparin itself, which have previously been reported [B. Mulloy, M.J. Forster, C. Jones, and D.B. Davies, Biochem. J., 293 (1993) 849-858]. The overall conformation of all the polysaccharides is shown to be similar, regardless of substitution pattern. The conformational equilibrium of the pyranose ring of iduronic acid residues in the polysaccharides has been monitored by the use of 13C NMR chemical shift temperature coefficients, and shown to be similar for all the modified heparins with the exception of N-desulphated re-N-acetylated heparin. Circular dichroism spectra of all the polysaccharides are reported, and their variations attributed to differences in the proportions of pyranose ring forms in the iduronate conformational equilibrium.

Conformational studies of synthetic peptides corresponding to the repetitive regions of the high molecular weight (HMW) glutenin subunits of wheat.

Circular dichroism and Fourier transform infrared spectra of three synthetic peptides based on the repeat motifs of the HMW subunits of glutenin showed that they formed β-turns when dissolved in trifluoroethanol, a solvent that favours ordered structures stabilized by hydrogen bonds. Peptide 1, based on the hexapeptide repeat motif, was predicted to form a type II β-turn, while peptides 2 and 3 (which span the functions between hexapeptide and nonapeptide motifs) were predicted to form overlapping turns of types II and I/III. These predictions are consistent with the structures determined in trifluoroethanol. In contrast all three peptides appeared to have unordered structures when dissolved in H 2 O or D 2 O. These results are discussed in relation to the conformations of the HMW subunits.

STRUCTURES, ASSAYS AND RECEPTORS FOR LOCUST ADIPOKINETIC HORMONES

This review is concerned mainly with the adipokinetic hormones (AKHs) of locusts: their molecular conformations, actions and functions and the development of microfiltration assays in vitro. The physiological significance of having multiple hormones with overlapping actions whose efficacy changes during development is discussed in relation to the possibility that these reflect variations in populations of receptors and/or the pharmacokinetics of the peptides. The involvement of second messengers in the transduction mechanism of AKHs is reviewed, and we describe hormone-induced changes of intracellular calcium in single dispersed fat body cells. The structure activity relationships of the three locust AKHs and a number of analogues with variations at the N- and C-termini are discussed. A number of areas are identified where there are gaps in our understanding of these hormones, and some of these will be the focus of our future research.

Divalent cation-sensitive pores formed by natural and synthetic melittin and by Triton X-100

Leakage of ions and low-molecular-weight metabolites from Lettre cells is induced by synthetic melittin, as effectively as by melittin isolated from bee venom; in each case leakage is inhibited by Ca2+, Zn2+ or H+. Inhibition of leakage by divalent cations is reversible in that Lettre cells incubated with melittin (or with Triton X-100) in the presence of inhibitory amounts of Zn2+, when freed of Zn2+ by EGTA or by centrifugation, begin to leak (in Zn2(+)-sensitive manner). Electrorotation of Lettre cells is altered by melittin, compatible with membrane permeabilization; melittin plus Zn2+ does not alter electrorotation until Zn2+ (and unbound melittin) are removed. Melittin or Triton X-100 added to calcein-loaded liposomes induces leakage of calcein; divalent cations inhibit. Energy transfer between liposome-associated melittin and 2-, 7- or 12-(9-anthroyloxy)stearate (AS) is maximal with 12-AS; addition of Zn2+ has little effect. Circular dichroism spectra of melittin plus liposomes are unaffected by Zn2+. These results show that the formation of divalent cation-sensitive pores is not dependent on the presence of endogenous membrane proteins and that the action of divalent cations is not by displacement of melittin (or Triton) from the lipid bilayer.

Chiroptical spectroscopic studies of polydiacetylenes

Abstract A new class of optically pure, chiral, soluble polydiacetylenes have been synthesized. These show both solvato- and thermo-chromism, with behaviours similar to those reported for the well-studied analogous achiral m -butoxycarbonylmethylurethane ( m BCMU) class of polydiacetylenes. Although it is now generally agreed that this chromic behaviour is a direct result of the order-disorder transformation of the dissolved polymer, there still remains disagreement about the conformation and state of aggregation of the various forms. The chiral structure of these new polymers allows these general questions to be addressed through the use of circular dichroism spectroscopy. We report measurements obtained on both solution and thin film samples. These have allowed us to characterize more precisely the various states involved.

Conformational properties of the prion octa-repeat and hydrophobic sequences

We have used circular dichroism to study synthetic peptides from two important regions of the prion protein: the N‐terminal octa‐repeat domain and a highly conserved hydrophobic section. Our results show that the octa‐repeat sequence in free solution can adopt a non‐random, extended conformation with properties similar to the poly‐l‐proline type II left‐handed helix. We also show that the conformation can be changed by temperature, organic solvents (e.g. acetonitrile) and on binding to phospholipid vesicles. We compared CD data from two peptides corresponding to the hydrophobic region between residues 106 and 136 which contained either methionine or valine at position 129. This variation represents a common polymorphism in humans which has been shown to influence predisposition towards iatrogenic and sporadic CJD. There was no detectable difference between the CD spectra of these peptides irrespective of the solvent conditions we used.

Left handed α‐helix formation by a bacterial peptide

The α‐helix is a common element of secondary structure in proteins and peptides. In eukaryotic organisms, which exclusively incorporate L‐amino acids into such molecules, stereochemical interactions make such α‐helices, invariably right‐handed. Pseudomonas tolaasii Paine is the causal organism of the economically significant brown blotch disease of the cultivated mushroom Agaricus bisporus (Lange) Imbach. P. tolaasii proceduces an extracellular lipodepsipeptide toxin, tolaasin, which causes the brown pitted lesions on the mushroom cap. Circular dichroism studies tolaasin in a membrane‐like environment indicate the presence of a left‐handed α‐helix, probably formed by a sequence of 7 D‐amino acids in the peptide. P. tolaasii represents the first reported example of an organism which has evolved the ability to biosynthesize a left‐handed α‐helix.

Spectroscopic characterisation of the reaction centre of photosystem II from higher plants

Two different photosystem II particles isolated from pea, a core complex and the D1/D2/Cyt b‐559 reaction centre (RC) complex, have been characterised by absorption, linear dichroism (LD) and circular dichroism (CD) spectroscopy. Only one carotenoid contributes to the LD of the reaction centre, in agreement with the biochemical analysis. This carotenoid is oriented parallel to the long axis of the reaction centre. The chlorophyll QY contribution to the LD is oriented perpendicular to the long axis of the reaction centre. The LD of the reaction centre carotenoid is reversed in the core complex. In addition, the contribution of a second carotenoid species can be observed. In the core, the two pools of carotenoid have a rather different orientation with respect to the membrane plane: one parallel, the other perpendicular. In addition, in the core the sign of the LD in the chlorophyll QY region is reversed and red‐shifted as compared to the reaction centre. These observations suggest that the reaction centre is oriented with its long axis perpendicular to the long axis of the PS II core and to the membrane plane. The circular dichroism CD of the reaction centre has intense peaks of opposite sign at 444 nm (negative) and 435 nm (positive), which we attribute to exciton coupling between Chl a molecules in the reaction centre. The RC has no CD in the range 460–650 nm; thus there is no exciton coupling between the carotenoid and the other pigments. The lack of CD in this region is consistent with the biochemical analysis of only one carotenoid per reaction centre. The larger core complex exhibits much weaker CD (per Chl). The CD in the QY absorption band indicates exciton coupling between chlorophyll molecules. The sign of the pair of peaks in this region is reversed in the core with respect to the reaction centre.

Resolution of conformational equilibria in linear peptides by circular dichroism in cryogenic solvents

The circular dichroism spectra of the synthetic peptide antigen, 209-222 of the surface glycoprotein of the rabies virus were recorded as a function of solvent composition and over the temperature range of +60 degrees C to -135 degrees C; beta-III and beta-II reverse turn conformations were found to exist in TFE/H2O (3:1) at room temperature and in ethanediol/H2O (2:1) below -110 degrees C respectively. Evidence, from comparison of observed and calculated spectra, is given to support the existence of a conformational equilibrium between a beta-II and a beta-III reverse turn. These data can serve as a basis for synthetic vaccine development and understanding the nature of polypeptide chain folding.