Under solution conditions where the native state is destabilized, the largely helical polypeptide hormone insulin readily aggregates to form amyloid fibrils with a characteristic cross-␤ structure. However, there is a lack of information relating the 4.8 Å ␤-strand repeat to the higher order assembly of amyloid fibrils. We have used cryo-electron microscopy (EM), combining single particle analysis and helical reconstruction, to characterize these fibrils and to study the three-dimensional (3D) arrangement of their component protofilaments. Low-resolution 3D structures of fibrils containing 2, 4, and 6 protofilaments reveal a characteristic, compact shape of the insulin protofilament. Considerations of protofilament packing indicate that the cross-␤ ribbon is composed of relatively flat ␤-sheets rather than being the highly twisted, ␤-coil structure previously suggested by analysis of globular protein folds. Comparison of the various fibril structures suggests that very small, local changes in ␤-sheet twist are important in establishing the long-range coiling of the protofilaments into fibrils of diverse morphology.U nder conditions that destabilize the native state, proteins can self-aggregate into insoluble, fibrillar assemblies (1-3). In the form of amyloid fibrils or fibril precursors, the proteins not only lack their original biological function but also may be harmful to organisms, causing pathologies such as Alzheimer's and prion diseases. Although amyloid precursor proteins do not share any sequence or structural homology, amyloid fibrils are typically unbranched, protease-resistant filaments approximately 100 Å in diameter and composed of Ϸ20-35 Å wide protofilaments, which are sometimes arranged around an electron lucent core (4, 5). Recently, several nonpathological proteins and short peptides have been shown to self-assemble into amyloid-like fibrils (6-9), leading to the suggestion that amyloid formation is a generic property of polypeptide chains (3, 10).The overall morphology of amyloid aggregates depends on the conditions in which fibrillogenesis takes place, and different fibril morphologies are often observed in the same preparation (7,(11)(12)(13)(14). Variable structures also are seen in ex vivo fibrils extracted from amyloidotic tissue (4, 15). The morphological variation seems to be caused by fibrils with a variable number and arrangement of protofilaments. X-ray fiber diffraction studies reveal a characteristic cross-␤ structure with ␤-strands of the precursor protein arranged perpendicular to, and ribbon-like ␤-sheets parallel to, the fibril axis (2, 16, 17). The ␤-strand repeat has also been directly visualized by cryo-EM (8). However, there is a lack of three-dimensional (3D) structural information on how the 4.8 Å ␤-strand repeat relates to the overall fibril assembly.The polypeptide hormone insulin has a mainly helical native structure, with its two polypeptide chains linked by two interchain and one intra-chain disulfide bonds (18). In vitro, insulin is readily converted to an inactiv...

Fourier transform infrared spectroscopy~FTIR!, circular dichroism~CD!, and electron microscopy~EM! have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like a-helical structure. On heating to 70 8C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from a-helical to b-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of ;450 Å. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.Keywords: CD; electron microscopy; fibril formation; FTIR; insulin; X-ray diffractionThe amyloid diseases, which include Alzheimer's disease, the spongiform encephalopathies and type II diabetes, are characterized by the abnormal self-assembly and deposition of proteinaceous material into insoluble ordered

Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism in Streptomyces roseosporus. A 128 kb region of S. roseosporus DNA was cloned and verified by heterologous expression in Streptomyces lividans to contain the daptomycin biosynthetic gene cluster (dpt). The cloned region was completely sequenced and three genes (dptA, dptBC, dptD) encoding the three subunits of an NRPS were identified. The catalytic domains in the subunits, predicted to couple five, six or two amino acids, respectively, included a novel activation domain and amino-acid-binding pocket for incorporating the unusual amino acid L-kynurenine (Kyn), three types of condensation domains and an extra epimerase domain (E-domain) in the second module. Novel genes (dptE, dptF ) whose products likely work in conjunction with a unique condensation domain to acylate the first amino acid, as well as other genes (dptI, dptJ) probably involved in supply of the non-proteinogenic amino acids L-3-methylglutamic acid and Kyn, were located next to the NRPS genes. The unexpected E-domain suggested that daptomycin would have D-Asn, rather than L-Asn, as originally assigned, and this was confirmed by comparing stereospecific synthetic peptides and the natural product both chemically and microbiologically.

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.

The substitution of alanines for the two cysteines which form thioether linkages to the haem group in cytochrome c 552 from Hydogenobacter thermophilus destabilises the native protein fold. The holo form of this variant slowly converts into a partially folded apo state that over prolonged periods of time aggregates into fibrillar structures. Characterisation of these structures by electron microscopy and thioflavin-T binding assays shows that they are amyloid fibrils. The data demonstrate that when the native state of this cytochrome is destabilised by loss of haem, even this highly K K-helical protein can form L L-sheet structures of the type most commonly associated with protein deposition diseases. ß

Daptomycin is a lipopeptide antibiotic produced by a nonribosomal peptide synthetase (NRPS) in Streptomyces roseosporus. The holoenzyme is composed of three subunits, encoded by the dptA, dptBC, and dptD genes, each responsible for incorporating particular amino acids into the peptide. We introduced expression plasmids carrying dptD or NRPS genes encoding subunits from two related lipopeptide biosynthetic pathways into a daptomycin nonproducing strain of S. roseosporus harboring a deletion of dptD. All constructs successfully complemented the deletion in trans, generating three peptide cores related to daptomycin. When these were coupled with incomplete methylation of 1 amino acid and natural variation in the lipid side chain, 18 lipopeptides were generated. Substantial amounts of nine of these compounds were readily obtained by fermentation, and all displayed antibacterial activity against gram-positive pathogens.

Sodium dodecyl sulphate (SDS), a detergent that mimics some characteristics of biological membranes, has been found to a¡ect signi¢cantly ¢bril formation by a peptide from human complement receptor 1. In aqueous solution the peptide is unfolded but slowly aggregates to form ¢brils. In sub-micellar concentrations of SDS the peptide is initially K K-helical but converts rapidly to a L L-sheet structure and large quantities of ¢brils form. In SDS above the critical micellar concentration the peptide adopts a stable K K-helical structure and no ¢brils are observed. These ¢ndings demonstrate the sensitivity of ¢bril formation to solution conditions and suggest a possible role for membrane components in amyloid ¢bril formation in living systems.

We have investigated the interactions between gramicidin and a model membrane composed of one phospholipid, dimyristoylphosphatidylcholine, as a function of the cosolubilization solvent and incubation time used in the sample preparation. Three organic solvents have been used; trifluoroethanol, a mixture of methanol/chloroform (1:1 v/v), and ethanol. Using Fourier transform infrared spectroscopy, we have demonstrated that the conformation adopted by gramicidin in the membrane is dependent upon the cosolubilization solvent used, and, only with trifluoroethanol, it is possible to incorporate gramicidin entirely as a beta 6.3-helix. Moreover, Raman spectroscopy results indicate that the orientation of the tryptophan side chains in gramicidin and their interaction with the hydrocarbon chains and the carbonyl groups of the lipids are also dependent on the cosolubilization solvent. On the other hand, the effect of the incorporation of gramicidin on the thermotropism of the lipid bilayer was found to be dependent upon the conformation of gramicidin in the lipid bilayers.