Fully synthetic immunogens. Part III. Synthesis of hinge-peptide/gastrin conjugates and their immunological properties.

As core molecule for the multiple attachment of antigenic peptides we have selected the human IgG1 hinge fragment 225-232/225'-232'. Two types of conjugates of this double-chain bis-cystinyl hinge-peptide were prepared i) by linking its C-termini to [NIe15]-human-little-gastrin-[2,17] and ii) by elongating the resulting hinge-peptide/[NIe15]-little-gastrin-[2-17] conjugate at the two N-termini with the human big-gastrin sequence 1-14 to produce the big-gastrin-[1-14]/hinge-peptide/little-gastrin-[2-17] conjugate. For the synthesis of these peptide structures both the route via the preformed double-chain bis-cystinyl peptide and the route via suitably protected monomeric bis-cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis-cysteinyl hinge-peptide 225-232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti-little-gastrin antibody titers comparable to those induced by the iso-1-cytochrome c/little-gastrin-[2-17] conjugate as carrier-hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor-like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little-gastrin-specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little-gastrin 2-17 and the big-gastrin 1-14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations.     

Fully synthetic immunogens. Part II. Studies on parallel dimerization of the human IgG1 hinge-fragment 225-232 with N- or C-terminal gastrin related extensions

The bis-cysteinyl hinge-fragment 225-232 of human IgG1 has been extended at the N- or C-terminus with Nle15-desamido-human-little-gastrin-[5-17] and Nle15-human-little-gastrin-[5-17]-NH2, respectively. Thermodynamically controlled air oxidation of the resulting bis-cysteinyl-peptides led to the predominant formation of the corresponding dimers in parallel alignment despite the incorporation of the immunoglobulin-unrelated gastrin-sequences. These surprising results confirm the high degree of structural information inherent in the hinge-sequence and its intrinsic tendency to fold into the correct structure in terms of cysteine pairings. This protein subdomain-the hinge-peptide-is therefore well suited as core molecule for the design of fully synthetic immunogens with multiple attachment of antigenic determinants.     

Synthesis of the bis-cystinyl-fragment 225-232/225'-232' of the human IgGl hinge region

In human IgGl the two heavy chains are crosslinked in the central portion of the molecule by two disulfide bridges forming a double chain bis-cystinyl cyclic peptide in parallel alignment. For our synthetic studies we have chosen the sequence portion 225-232/225'-232', i.e. [H-Thr-C1ys-Pro-Pro-C1ys-Pro-Ala-Pro-OH]2. By the use of a combination of the S-tert.-butylthio and the S-acetamidomethyl groups selective cysteine pairings in two successive steps produced the hinge hexadecapeptide in parallel and antiparallel alignment as homogeneous and well characterized compounds. Thiol disulfide interchange experiments on the antiparallel dimer led to over 90% conversion to the parallel isomer. Similarly random air-oxidation was found to generate again mainly the parallel dimer, thus strongly suggesting that this sequence portion contains sufficient structural information for a correct assembly of the two heavy chains of immunoglobulins without decisive contribution of a protein disulfide isomerase.     

Evidence for a C-terminal structural motif in gastrin and its bioactive fragments in membrane mimetic media

The conformational preferences of human little gastrin, [Nle(15)] gastrin-17, and its short analogues, gastrin-4 and [beta-Ala(1)] gastrin-5, which include the C-terminal tetrapeptide sequence Trp-Met-Asp-Phe-NH(2) crucial for gastrin bioactivity, were determined by NMR spectroscopy in aqueous solutions of zwitterionic dodecylphosphocholine micelles. Backbone HN chemical shift temperature variance, Halpha chemical shift deviations and complex non-sequential NOE patterns pointed to the C-terminal of [Nle(15)] gastrin-17 adopting an ordered conformation. Distance geometry calculations and NOE-restrained molecular dynamics simulations in membrane mimetic solvent boxes of decane and water indicated the C-terminal tetrapeptide sequence of all three peptides adopted a similar, well defined structure, with a general type IV beta-turn observed for all three peptides. The conformation of [Nle(15)] gastrin-17 consisted of two short helices between Leu(5)-Glu(9) and Ala(11)-Trp(14), with the one helix terminating in a type I beta-turn spanning Gly(13)-Asp(16). The experimental evidence and conformational characteristics of the three peptides in micellar media support a membrane-associated mechanism of receptor recognition and activation for the gastrin hormone family and furthermore point to a possible biologically relevant structural motif for gastrin activity.     

The reactivity and oxidation pathway of cysteine 232 in recombinant human alpha 1-antitrypsin

Oxidative damage to the sulfur-containing amino acids, methionine and cysteine, is a major concern in biotechnology and medicine. alpha1-Antitrypsin, which is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors, contains nine methionines and a single cysteine in its primary sequence. Although it is known that methionine oxidation in the protein active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of its unpaired thiol, Cys-232. In this study, the thiol-modifying reagent NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole) was used to label peroxide-modified alpha1-antitrypsin and demonstrate that the Cys-232 in vitro oxidation pathway begins with a stable sulfenic acid intermediate and is followed by the formation of sulfinic and cysteic acid in successive steps. pH-dependent reactivity with hydrogen peroxide showed that Cys-232 has a pK(a) of 6.86 +/- 0.05, a value that is more than 1.5 pH units lower than that of a typical protein thiol. pH-induced conformational changes in the region surrounding Cys-232 were also examined and indicate that mildly acidic conditions induce a conformation that enhances Cys-232 reactivity. In summary, this work provides new insights into alpha1-antitrypsin reactivity in oxidizing environments and shows that a unique structural environment renders its unpaired thiol, Cys-232, its most reactive amino acid.     

Folding of thermolysin fragments. Correlation between conformational stability and antigenicity of carboxyl-terminal fragments

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH-terminal fragments 121-316, 206-316 and 225(226)-316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two-fragment complex composed of fragments 5-224(225) and 225(226)-316. In aqueous solution at neutral pH, all the COOH-terminal fragments attain a native-like conformation, as judged both by the content of secondary structure deduced from far-ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH-terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn X HCl). The phase transition curves were analyzed for Tm (temperature of half-denaturation) and Gibbs free energies (delta GD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)-316 less than or equal to 206-316 less than 121-316 less than thermolysin S less than thermolysin. The ranking of delta GD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C-labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206-316 of native thermolysin indicate that the COOH-terminal fragments adopt native-like conformations which are in equilibrium with non-native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)-316, to 206-316, to 121-316. Fragment 225(226)-316, when combined with fragment 5-224(225) in the thermolysin S complex, adopts a more stable native-like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH-terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.     

Conformational studies of a short linear peptide corresponding to a major conserved neutralizing epitope of human respiratory syncytial virus fusion glycoprotein

The conformational properties of a 21-residue peptide, corresponding to amino acids 255 to 275 (F255-275) of the human respiratory syncytial virus fusion (F) glycoprotein, have been studied by CD and nmr spectroscopy. This peptide includes residues 262, 268, and 272 of the F polypeptide that are essential for integrity of most epitopes that mapped into a major antigenic site of the F molecule. CD data indicate that F255-275 adopts a random coil conformation in aqueous solution at low peptide concentrations. However, as the concentration of peptide is increased, a higher percentage of peptide molecules adopts an organized structure. This effect can be more easily observed when trifluoroethanol (30%) is added to peptide solutions, giving rise to CD spectra that resemble those of α-helix structures. These conformational changes were confirmed by nmr spectroscopy. The nuclear Overhauser effects observed in 30% trifluoroethanol/water together with the conformational H_α chemical shift data allowed us to propose a structural model of helix-loop-helix for the peptide in solution. In addition, these helical regions contain the amino acid residues essential for epitope integrity in the native F molecule. These results give new insights into the antigenic structure of the respiratory syncytial virus F glycoprotein. © 1996 John Wiley & Sons, Inc.     

Conformational aspects of gastrin-related peptides: A circular dichroism study

The conformational properties of a series of gastrin-related peptides in aqueous solution and in 2,2,2-trifluoroethanol (TFE) have been investigated by CD measurements. In aqueous solution the peptides Leu³²-HG-34 (human big gastrin), Nle¹⁵-HG-17 (human little gastrin), and Nle¹¹-HG-13 assume a random-coil structure in the pH range 3–7. In TFE the three hormones fold into partially ordered structures, consisting of mixtures of α-helix, β-form and random coil. Comparison with the CD properties of the shorter gastrin peptides HG-4 (tetragastrin), N^α-Boc-HG-5 (pentagastrin), and HG-7 (heptagastrin) indicates that the biologically important C-terminal sequence Trp-Met-Asp-Phe-NH₂ in TFE does not maintain the same geometry upon elongation of the chain at the N-terminus from 4 to 34 residues. Thus, the various conformations in solution of the gastrin peptides examined do not provide a structural explanation for their very similar biological activity. Therefore, we hypothesize that the C-terminal tetrapeptide amide folds into an “active” structure only upon interaction with the receptor.     

Conformational studies of human [15-2-aminohexanoic acid]little gastrin in sodium dodecyl sulfate micelles by 1H NMR

Human little gastrin is a 17 amino acid peptide that adopts a random conformation in water and an ordered structure in sodium dodecyl sulfate (SDS) micelles as well as in trifluoroethanol (TFE). The circular dichroism spectra in these two media have the same shape, indicative of a similar preferred conformation [Mammi, S., Mammi, N. J., Foffani, M. T., Peggion, E., Moroder, L., & Wünsch, E. (1987) Biopolymers 26, S1-S10]. We describe here the assignment of the proton NMR resonances and the conformational analysis of [Ahx15]little gastrin in SDS micelles. Two-dimensional correlation techniques form the basis for the assignment. The conformational analysis utilized NOE's, NH to C alpha H coupling constants, and the temperature coefficients of the amide chemical shifts. The NMR data indicate a helical structure in the N-terminal portion of the peptide. These results are compared with the conformation that we recently proposed for a minigastrin analogue (fragment 5-17 of [Ahx15]little gastrin) in TFE.     

Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells

Gastrin17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met15]gastrin17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin17gly or gastrin17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle15]gastrin2,17gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of gastrin17gly to DLD-1 cells was 2.1+/-0.4 microM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.     

The mechanisms of 232Th and of Ce (III) toxic action on Chlorella vulgaris Beijer. Estimation of 232Th radiation impact contribution into toxic effect induced

232Th and Ce (III) toxic effects and its modifications with caffeine and D,L-buthionine-(S,R)-sulphoximine on Chlorella vulgaris Beijer were studied using an optical density measure after 24 hours growth. Concentrations of 232Th or of Ce--toxic effect relationship were shown to be nonlinear. In the first (nontoxic) concentration range (for Ce 0.036-1.642 micromol/L and for 232Th 0.001-1.551 micromol/L) algae biomass production registered by optical density do not significantly differ from the control one. In the second (toxic) concentration range dose-effect relationship for 232Th is characterized with quadratic dependence and in the case of Ce--with exponential dependence. 232Th radiation component contribution into effect observed is appeared as induction of DNA damages additional to spontaneous at the radionuclide concentration (equal to 0.345 micromol/L) that is three times lower than in case of the non-radioactive chemical analog Ce (1.071 micromol/L).     

Structural and physicochemical analysis of the reaction between the anti-lysozyme antibody D1.3 and the anti-idiotopic antibodies E225 and E5.2

The reaction between the mouse (BALB/c) anti-idiotiopic monoclonal antibodies E225 and E5.2 and idiotopes on the (BALB/c) anti-lysozyme monoclonal antibody D1.3 has been characterized by titration calorimetry, by equilibrium sedimentation and by the determination of binding association and dissociation rates. The reaction between E5.2 and D1.3 is driven by a large negative enthalpy and its rate and equilibrium association constants are comparable to those observed in other antigen–antibody reactions. In contrast, the reaction between E225 and D1.3 is entropically driven and characterized by slow association kinetic (1 × 10³ M^?1 sec^?1) and a resulting low equilibrium constant (K_a = 2 × 10⁵M ^?1). A correlation of these properties with the three-dimensional structure of the Fab225-FabD1.3 complex, previously determined by X-ray diffraction methods to 2.5 Å resolution, indicates that conformational changes of several D1.3 contacting residues, located in its complementarity determining regions, may explain these features of the reaction.     

Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor

The conformational features of a conjugate of the C-terminus of human gastrin (HG[11-17]), the shortest gastrin sequence retaining biological function, with beta-cyclodextrin ([Nle(15)]-HG[11-17]-betaCD) were determined by NMR spectroscopy in an aqueous solution of dodecylphosphocholine (DPC) micelles. The peptide-betaCD conjugate displays a binding affinity and activation profile comparable to those of HG[11-17] at the cholecysokinin 2 (CCK(2)) receptor, the G protein-coupled receptor responsible for the gastrointestinal function of gastrin. The structure of the peptide consisted of a well-defined beta-turn between Gly(13) and Asp(16) of gastrin. The structural preferences of [Nle(15)]-HG[11-17]-betaCD in DPC micelles and the 5-doxylstearate-induced relaxation of the (1)H NMR resonances support a membrane-associated receptor recognition mechanism. Addition of [Nle(15)]-HG[11-17]-betaCD to the third extracellular loop domain of the CCK(2) receptor, CCK(2)-R(352-379), generated a number of intermolecular nuclear Overhauser enhancements (NOEs) and chemical shift perturbations. NOE-restrained MD simulations of the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex produced a topological orientation in which the C-terminus was located in a shallow hydrophobic pocket near the confluence of TM2 and -3. Despite the steric bulk and physicochemical properties of betaCD, the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex is similar to the CCK-8-CCK(2)-R complex determined previously, providing insight into the mode of ligand binding and the role of electrostatic interactions.     

14-3-3zeta C-terminal stretch changes its conformation upon ligand binding and phosphorylation at Thr232

14-3-3 proteins are abundant binding proteins involved in many biologically important processes. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. In this work, we studied the conformational changes of 14-3-3 C-terminal stretch, a region implicated in playing a role in the regulation of 14-3-3. Time-resolved fluorescence and molecular dynamics were used to investigate structural changes of the C-terminal stretch induced by phosphopeptide binding and phosphorylation at Thr232, a casein kinase I phosphorylation site located within this region. A tryptophan residue placed at position 242 was exploited as an intrinsic fluorescence probe of the C-terminal stretch dynamics. Other tryptophan residues were mutated to phenylalanine. Time-resolved fluorescence measurements revealed that phosphopeptide binding changes the conformation and increases the flexibility of 14-3-3zeta C-terminal stretch, demonstrating that this region is directly involved in ligand binding. Phosphorylation of 14-3-3zeta at Thr232 resulted in inhibition of phosphopeptide binding and suppression of 14-3-3-mediated enhancement of serotonin N-acetyltransferase activity. Time-resolved fluorescence of Trp242 also revealed that phosphorylation at Thr232 induces significant changes of the C-terminal stretch conformation. In addition, molecular dynamic simulations suggest that phosphorylation at Thr232 induces a more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. These results indicate that the conformation of the C-terminal stretch plays an important role in the regulation of 14-3-3 binding properties.     

Cation binding sites on actin: a structural relationship between antigenic epitopes and cation exchange

Divalent cations such as Mg2+ and Ca2+, which bind specifically to actin, induce conformational changes that affect its antigenic structure. The distribution of antigenic epitopes on the sequence shows that these structural modifications involve epitopes related to monomer-monomer interfaces. In the N-terminal part, the 1-7 acidic extremity is not affected, in contrast with sequence 18-28. The ability of polycations such as diamine to modify the actin structure at concentrations below 0.1 microM strengthens the hypothesis that in vivo these compounds act locally and specifically on actin polymerization.     

Replacement of the Human Topoisomerase Linker Domain with the Plasmodial Counterpart Renders the Enzyme Camptothecin Resistant

A human/plasmodial hybrid enzyme, generated by swapping the human topoisomerase IB linker domain with the corresponding domain of the Plasmodium falciparum enzyme, has been produced and characterized. The hybrid enzyme displays a relaxation activity comparable to the human enzyme, but it is characterized by a much faster religation rate. The hybrid enzyme is also camptothecin resistant. A 3D structure of the hybrid enzyme has been built and its structural-dynamical properties have been analyzed by molecular dynamics simulation. The analysis indicates that the swapped plasmodial linker samples a conformational space much larger than the corresponding domain in the human enzyme. The large linker conformational variability is then linked to important functional properties such as an increased religation rate and a low drug reactivity, demonstrating that the linker domain has a crucial role in the modulation of the topoisomerase IB activity.     

Photosensitized oxidation of elastase. Selective modification of the tryptophyl residues

Spectral studies demonstrated that acidic pH values induce a two-step denaturation of porcine elastase, the first conformational transition occuring over the pH range 4.2–3.8, the second between pH 3.3 and 2.9. The proflavine-sensitized photooxidation of elastase in its native state, as well as in its denatured conformations, allowed us to isolate elastase derivatives selectively modified at given tryptophyl residues, hence to draw reliable conclusions about their degree of burial inside the protein matrix and their functional and conformational role. In particular, tryptophan-26 and -164 are located at the surface of the protein molecule, and their oxidation to N-formylkynurenine has no appreciable effect on the elastolytic activity and three-dimensional geometry of elastase. Tryptophan-83 is partially shielded from the aqueous environment; its modification affects only slightly the enzymic efficiency, while the tertiary structure of the protein perhaps increases its rigidity. Tryptophan-12 must be largely buried in internal regions, since its photooxidation is possible only after the native elastase structure has been extensively randomized; its indole ring appears to be of critical importance for the enzymic activity and the conformational stability of elastase. Finally, our data suggest that tryptophan-39, -132, and -232 are deeply buried; consequently, we failed to achieve the specific or preferential modification of these residues.     

Glycine-extended gastrin exerts growth-promoting effects on human colon cancer cells.

BACKGROUND: Since human colon cancers often contain significant quantities of progastrin-processing intermediates, we sought to explore the possibility that the biosynthetic precursor of fully processed amidated gastrin, glycine-extended gastrin, may exert trophic effects on human colonic cancer cells. MATERIALS AND METHODS: Binding of radiolabeled glycine-extended and amidated gastrins was assessed on five human cancer cell lines: LoVo, HT 29, HCT 116, Colo 320DM, and T 84. Trophic actions of the peptides were assessed by increases in [3H]thymidine incorporation and cell number. Gastrin expression was determined by northern blot and radioimmunoassay. RESULTS: Amidated gastrin did not bind to or stimulate the growth of any of the five cell lines. In contrast, saturable binding of radiolabeled glycine-extended gastrin was seen on LoVo and HT 29 cells that was not inhibited by amidated gastrin (10(-6) M) nor by a gastrin/CCKB receptor antagonist (PD 134308). Glycine-extended gastrin induced a dose-dependent increase in [3H]thymidine uptake in LoVo (143 +/- 8% versus control at 10(-10) M) and HT 29 (151 +/- 11% versus control at 10(-10) M) cells that was not inhibited by PD 134308 or by a mitogen-activated protein (MAP) or ERK kinase (MEK) inhibitor (PD 98509). Glycine-extended gastrin did stimulate jun-kinase activity in LoVo and HT 29 cells. The two cell lines expressed the gastrin gene at low levels and secreted small amounts of amidated gastrin and glycine-extended gastrin into the media. CONCLUSIONS: Glycine-extended gastrin receptors are present on human colon cancer cells that mediate glycine-extended gastrin's trophic effects via a MEK-independent mechanism. This suggests that glycine-extended gastrin and its novel receptors may play a role in colon cancer cell growth.     

Solution structure of human growth hormone-releasing factor fragment (1-29) by CD: characteristic conformational change on phospholipid membrane

The conformations of synthetic human growth hormone-releasing factor fragment (1-29) in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol liposome as well as in aqueous 2,2,2-trifluoroethanol solution were investigated by CD spectroscopy. The secondary structure of the peptide in each solution was analyzed by two methods. Both results show that the peptide has an unordered structure in the aqueous solution, whereas it folds into helical structure in the aqueous alcohol and in the phospholipid solution. In addition, although the peptide exists as almost complete helix in the 50 vol% aqueous alcohol (80-90% helicity), it does not reach full helicity even in the solution containing excess amount of phospholipid liposome (maximum 65-70% helicity). The conformational difference is explained by the characteristic amphipathy of the peptide, i.e., the necessity to twist the separated amphipathic helical parts in the interaction with the phospholipid membrane probably makes the helicity of the peptide decrease.     

Molecular determinants of amyloid deposition in Alzheimer's disease: conformational studies of synthetic beta-protein fragments

The amyloid beta-protein (1-42) is a major constituent of the abnormal extracellular amyloid plaque that characterizes the brains of victims of Alzheimer's disease. Two peptides, with sequences derived from the previously unexplored C-terminal region of the beta-protein, beta 26-33 (H2N-SNKGAIIG-CO2H) and beta 34-42 (H2N-LMVGGVVIA-CO2H), were synthesized and purified, and their solubility and conformational properties were analyzed. Peptide beta 26-33 was found to be freely soluble in water; however, peptide beta 34-42 was virtually insoluble in aqueous media, including 6 M guanidinium thiocyanate. The peptides formed assemblies having distinct fibrillar morphologies and different dimensions as observed by electron microscopy of negatively stained samples. X-ray diffraction revealed that the peptide conformation in the fibrils was cross-beta. A correlation between solubility and beta-structure formation was inferred from FTIR studies: beta 26-33, when dissolved in water, existed as a random coil, whereas the water-insoluble peptide beta 34-42 possessed antiparallel beta-sheet structure in the solid state. Solubilization of beta 34-42 in organic media resulted in the disappearance of beta-structure. These data suggest that the sequence 34-42, by virtue of its ability to form unusually stable beta-structure, is a major contributor to the insolubility of the beta-protein and may nucleate the formation of the fibrils that constitute amyloid plaque.