Analysis of Mitochondrial Membrane Fusion GTPase OPA1 Expressed by the Silkworm Expression System.

Mitochondria are highly dynamic organelles, which move and fuse to regulate their shape, size, and fundamental function. The dynamin-related GTPases play a critical role in mitochondrial membrane fusion. In vitro reconstitution of membrane fusion using recombinant proteins and model membranes is quite useful in elucidating the molecular mechanisms underlying membrane fusion and to identify the essential elements involved in fusion. However, only a few reconstituting approaches have been reported for mitochondrial fusion machinery due to the difficulty of preparing active recombinant mitochondrial fusion GTPases. Recently, we succeeded in preparing a sufficient amount of recombinant OPA1 involved in mitochondrial inner membrane fusion using a BmNPV bacmid-silkworm expression system. In this chapter, we describe the method for the expression and purification of a membrane-anchored form of OPA1 and liposome-based in vitro reconstitution of membrane fusion.

Relationship between OPA1 and cardiolipin in mitochondrial inner-membrane fusion.

Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. The large GTPase optic atrophy 1 (OPA1) is identified as a core component of inner membrane (IM) fusion. OPA1 exists as the membrane-anchored L-OPA1 and the proteolytically cleavage soluble S-OPA1. Recently, we showed that OPA1 and mitochondria-localized lipid cardiolipin (CL) cooperate in heterotypic IM fusion [Ban et al., Nat. Cell Biol. 19 (2017) 856-863]. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 and S-OPA1 expressed in silkworm and found that L-OPA1 on one side of the membrane and CL on the other side were sufficient for mitochondrial fusion. L-OPA1 is the major fusion-prone factor in heterotypic fusion. However, the role of S-OPA1 remains unknown as S-OPA1 promoted L-OPA1-dependent heterotypic membrane fusion and homotypic CL-containing membrane fusion, but S-OPA1 alone was not sufficient for heterotypic membrane fusion. L-OPA1- and CL-mediated heterotypic mitochondrial fusion was confirmed in living cells, but tafazzin (Taz1), the causal gene product of Barth syndrome, was not essential for mitochondrial fusion. Taz1-dependent CL maturation might have other roles in the remodeling of mitochondrial DNA nucleoids.

Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin.

Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. Optic atrophy 1 (OPA1) is an essential GTPase protein for both mitochondrial inner membrane (IM) fusion and cristae morphology. Under mitochondria-stress conditions, membrane-anchored L-OPA1 is proteolytically cleaved to form peripheral S-OPA1, leading to the selection of damaged mitochondria for mitophagy. However, molecular details of the selective mitochondrial fusion are less well understood. Here, we showed that L-OPA1 and cardiolipin (CL) cooperate in heterotypic mitochondrial IM fusion. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 protein expressed in silkworm, and found that L-OPA1 on one side of the membrane and CL on the other side are sufficient for fusion. GTP-independent membrane tethering through L-OPA1 and CL primes the subsequent GTP-hydrolysis-dependent fusion, which can be modulated by the presence of S-OPA1. These results unveil the most minimal intracellular membrane fusion machinery. In contrast, independent of CL, a homotypic trans-OPA1 interaction mediates membrane tethering, thereby supporting the cristae structure. Thus, multiple OPA1 functions are modulated by local CL conditions for regulation of mitochondrial morphology and quality control.

Cell-free mitochondrial fusion assay detected by specific protease reaction revealed Ca2+ as regulator of mitofusin-dependent mitochondrial fusion.

Mitochondrial dynamic by frequent fusion and fission have important roles in various cellular signalling processes and pathophysiology in vivo. However, the molecular mechanisms that regulate mitochondrial fusion, especially in mammalian cells, are not well understood. Accordingly, we developed a novel biochemical cell-free mitochondrial fusion assay system using isolated human mitochondria. We used a protease and its specific substrate that are essential for yeast autophagy; Atg4 protease is required for maturation and the de-conjugation of the ubiquitin-like modifier Atg8. Atg4-FLAG and Atg8-GFP were separately expressed in the mitochondrial matrix of HeLa cells. Isolated mitochondria were then mixed and packed in the presence of energy regeneration mix. Immunoblotting with an anti-GFP antibody revealed Atg8 processing, suggesting that the double membranes of isolated mitochondria were indeed fused. The mitochondrial fusion reaction required GTP hydrolysis, mitochondrial membrane potential and intact outer membrane proteins containing two mitofusin isoforms. Using this assay, we searched for stimulators of mitochondrial fusion and found that rabbit reticulocyte lysate and Ca2+ chelator EGTA stimulate mitochondrial fusion. This novel cell-free assay system using isolated human mitochondria is simple, sensitive and reproducible; thus, it is useful for screening proteins and molecules that modulate mitochondrial fusion.

OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation.

The dynamin-related GTPase OPA1 is mutated in autosomal dominant optic atrophy (DOA) (Kjer type), an inherited neuropathy of the retinal ganglion cells. OPA1 is essential for the fusion of the inner mitochondrial membranes, but its mechanism of action remains poorly understood. Here we show that OPA1 has a low basal rate of GTP hydrolysis that is dramatically enhanced by association with liposomes containing negative phospholipids such as cardiolipin. Lipid association triggers assembly of OPA1 into higher order oligomers. In addition, we find that OPA1 can promote the protrusion of lipid tubules from the surface of cardiolipin-containing liposomes. In such lipid protrusions, OPA1 assemblies are observed on the outside of the lipid tubule surface, a protein-membrane topology similar to that of classical dynamins. The membrane tubulation activity of OPA1 is suppressed by GTPgammaS. OPA1 disease alleles associated with DOA display selective defects in several activities, including cardiolipin association, GTP hydrolysis and membrane tubulation. These findings indicate that interaction of OPA1 with membranes can stimulate higher order assembly, enhance GTP hydrolysis and lead to membrane deformation into tubules.

Branching in amyloid fibril growth.

Using the peptide hormone glucagon and Abeta(1-40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35-40 degrees . Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Abeta(1-40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and Abeta(1-40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth.

Destruction of amyloid fibrils of a beta2-microglobulin fragment by laser beam irradiation.

To understand the mechanism by which amyloid fibrils form, we have been making real-time observations of the growth of individual fibrils, using total internal fluorescence microscopy combined with an amyloid-specific fluorescence dye, thioflavin T (ThT). At neutral pH, irradiation at 442 nm with a laser beam to excite ThT inhibited the fibril growth of beta(2)-microglobulin (beta2-m), a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Examination with a 22-residue K3 fragment of beta2-m showed that the inhibition of fibril growth and moreover the destruction of preformed fibrils were coupled with the excitation of ThT. Several pieces of evidence suggest that the excited ThT transfers energy to ground state molecular oxygen, producing active oxygen, which causes various types of chemical modifications. The results imply a novel strategy for preventing the deposition of amyloid fibrils and for destroying preformed amyloid deposits.

Structure, formation and propagation of amyloid fibrils.

Amyloid fibrils have been a critical subject in recent studies of proteins since they are associated with the pathology of more than 20 serious human diseases. Moreover, a variety of proteins and peptides not related to diseases are able to form amyloid fibrils or amyloid-like structures, implying that amyloid formation is a generic property of polypeptides. Although understanding the structure and formation of amyloid fibrils is crucial, due to the extremely high molecular weight and insolubility of amyloid fibrils, most of the conventional techniques available for soluble proteins are not directly applicable to these fibrils. However, structural studies using solid-state NMR have shown that the basic motif of amyloid fibrils is a beta-strand-loop-beta-strand conformation often in a parallel beta-sheet assembly. From the hydrogen/deuterium exchange of amide protons, amyloid fibrils have been shown to be stabilized by an extensive network of hydrogen bonds substantiating beta-sheets. Our approach using total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye, enabled monitoring fibril growth in real-time at single fibril level. On the basis of these various approaches, increasingly convincing models of amyloid structures, their formation and propagation are emerging.

Biotin-containing phospholipid vesicle layer formed on self-assembled monolayer of a saccharide-terminated alkyl disulfide for surface plasmon resonance biosensing.

We describe a technique to form a biotin-containing phospholipid vesicle layer on a self-assembled monolayer (SAM) deposited on a gold surface to immobilize biotinylated receptor proteins for a surface plasmon resonance (SPR) biosensor. The adsorption of vesicle of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was examined by SPR on the SAMs of dithiobis(1-deoxy-glucitol-1-carbamoyl pentane) (DDGP), 11-mercaptoundecanoic acid, 11-mercaptoundecanol, 11-amino-1-undecanethiol, and 12-mercaptododecane, and it was found that the DOPC vesicle rapidly adsorbed on the DDGP SAM to achieve the highest coverage of the surface. By quartz crystal microbalance with dissipation monitoring (QCM-D), the DOPC layer formed on the DDGP SAM was shown to be a vesicle layer, in which intact DOPC vesicles physisorbed on the SAM surface. To immobilize a biotinylated receptor protein, one of three biotinylated phospholipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotin-DOPE), N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-X-DHPE) and N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-DHPE), was mixed with DOPC to form a biotin-containing vesicle layer on the DDGP SAM. A comparative binding study of NeutrAvidin and the biotin-containing vesicle layers showed that the use of biotin-X-DHPE achieved the most rapid immobilization of NeutrAvidin on the vesicle layer at the highest surface density. Furthermore, biotinylated protein A, as a receptor protein, could be immobilized through NeutrAvidin on the vesicle layer containing DOPC and biotin-X-DHPE, and its reaction with immunoglobulin G, as an analyte, was successfully observed by SPR. The results demonstrate that the biotin-containing vesicle layer on the DDGP SAM must be a useful component for SPR biosensor surfaces.

Visualization and classification of amyloid beta supramolecular assemblies.

Deposition of amyloid beta (Abeta) fibrils has been suggested to play a central role in Alzheimer's disease. In clarifying the mechanism by which fibrils form and moreover in developing new treatments for amyloidosis, direct observation is important. Focusing on the interactions with surfaces at the early stages, we studied the spontaneous formation of Abeta(1-40) fibrils on quartz slides, monitored by total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye. Self-assembly of Abeta(1-40), accelerated by a low concentration of sodium dodecyl sulfate, produced various remarkable amyloid assemblies. Densely packed spherulitic structures with radial fibril growth were typically observed. When the packing of fibrils was coarse, extremely long fibrils often protruded from the spherulitic cores. In other cases, a large number of wormlike fibrils were formed. Transmission electron microscopy and atomic force microscopy revealed relatively short and straight fibrillar blocks associated laterally without tight interaction, leading to random-walk-like fibril growth. These results suggest that, during spontaneous fibrillation, the nucleation occurring in contact with surfaces is easily affected by environmental factors, creating various types of nuclei, and hence variations in amyloid morphology. A taxonomy of amyloid supramolecular assemblies will be useful in clarifying the structure-function relationship of amyloid fibrils.

Lipocalin-type prostaglandin D synthase/beta-trace is a major amyloid beta-chaperone in human cerebrospinal fluid.

The conformational change in amyloid beta (Abeta) peptide from its monomeric form to aggregates is crucial in the pathogenesis of Alzheimer's disease (AD). In the healthy brain, some unidentified chaperones appear to prevent the aggregation of Abeta. Here we reported that lipocalin-type prostaglandin D synthase (L-PGDS)/beta-trace, the most abundant cerebrospinal fluid (CSF) protein produced in the brain, was localized in amyloid plaques in both AD patients and AD-model Tg2576 mice. Surface plasmon resonance analysis revealed that L-PGDS/beta-trace tightly bound to Abeta monomers and fibrils with high affinity (K(D) = 18-50 nM) and that L-PGDS/beta-trace recognized residues 25-28 in Abeta, which is the key region for its conformational change to a beta-sheet structure. The results of a thioflavin T fluorescence assay to monitor Abeta aggregation disclosed that L-PGDS/beta-trace inhibited the spontaneous aggregation of Abeta (1-40) and Abeta (1-42) within its physiological range (1-5 microM) in CSF. L-PGDS/beta-trace also prevented the seed-dependent aggregation of 50 microM Abeta with K(i) of 0.75 microM. Moreover, the inhibitory activity toward Abeta (1-40) aggregation in human CSF was decreased by 60% when L-PGDS/beta-trace was removed from the CSF by immunoaffinity chromatography. The deposition of Abeta after intraventricular infusion of Abeta (1-42) was 3.5-fold higher in L-PGDS-deficient mice and reduced to 23% in L-PGDS-overexpressing mice as compared with their wild-type levels. These data indicate that L-PGDS/beta-trace is a major endogenous Abeta-chaperone in the brain and suggest that the disturbance of this function may be involved in the onset and progression of AD. Our findings may provide a diagnostic and therapeutic approach for AD.

Direct observation of amyloid growth monitored by total internal reflection fluorescence microscopy.

Most morphological investigations of amyloid fibrils have been performed with various microscopic methods. Among them, direct observation of fibril growth is possible using atomic force microscopy and fluorescence microscopy. Direct observation provides information about the rate and direction of growth at the single fibril level, which cannot be obtained from averaged ensemble measurements. In this chapter, we describe a new technique for the direct observation of amyloid fibril growth using total internal reflection fluorescence microscopy (TIRFM) combined with amyloid-specific thioflavin T (ThT) fluorescence. TIRFM has been developed to monitor single molecules by effectively reducing the background fluorescence in an evanescent field. One of the advantages of TIRFM is that one can selectively monitor fibrils lying along a glass slide, so that one can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent fibril growth of amyloid beta (1-40). The fibril growth was a highly cooperative process, with the fibril ends extending at a constant rate. Because ThT binding is common to all amyloid fibrils, the present method will have general applicability to the real-time analysis of amyloid fibrils.

Direct observation of amyloid fibril growth, propagation, and adaptation.

Amyloid fibrils form through nucleation and growth. To clarify the mechanism involved, direct observations of both processes are important. First, seed-dependent fibril growth of beta2-microglobulin (beta2-m) and amyloid beta peptide was visualized in real time at the single fibril level using total internal reflection fluorescence microscopy combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. Second, using atomic force microscopy, ultrasonication-induced formation of beta2-m fibrils was shown, indicating that ultrasonication is useful to accelerate the nucleation process. Third, with the proteolytic fragment of beta2-m, propagation and a transformation of fibril morphology was demonstrated. These direct observations indicate that template-dependent growth and structural diversity are key factors determining the structure and function of amyloid fibrils.

Real-time and single fibril observation of the formation of amyloid beta spherulitic structures.

In Alzheimer disease, amyloid beta, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 microm on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

AlphaB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid beta-peptide and beta2-microglobulin.

AlphaB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of alphaB-crystallin on the fibril growth of the Abeta (amyloid beta)-peptides Abeta-(1-40) and Abeta-(1-42). alphaB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Abeta-(1-40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy. Comparison of the activity of some mutants and chimaeric alpha-crystallins in preventing Abeta-(1-40) fibril growth with their previously reported chaperone ability in preventing dithiothreitol-induced aggregation of insulin suggests that there might be both common and distinct sites of interaction on alpha-crystallin involved in the prevention of amorphous aggregation of insulin and fibril growth of Abeta-(1-40). alphaB-crystallin also prevents the spontaneous fibril formation (without externally added seeds) of Abeta-(1-42), as well as the fibril growth of Abeta-(1-40) when seeded with the Abeta-(1-42) fibril seed. Sedimentation velocity measurements show that alphaB-crystallin does not form a stable complex with Abeta-(1-40). The mechanism by which it prevents the fibril growth differs from the known mechanism by which it prevents the amorphous aggregation of proteins. alphaB-crystallin binds to the amyloid fibrils of Abeta-(1-40), indicating that the preferential interaction of the chaperone with the fibril nucleus, which inhibits nucleation-dependent polymerization of amyloid fibrils, is the mechanism that is predominantly involved. We found that alphaB-crystallin prevents the fibril growth of beta2-microglobulin under acidic conditions. It also retards the depolymerization of beta2-microglobulin fibrils, indicating that it can interact with the fibrils. Our study sheds light on the role of small heat-shock proteins in protein conformational diseases, particularly in Alzheimer's disease.

Structural observation of complexes of FGF-2 and regioselectively desulfated heparin in aqueous solutions.

In order to clarify the mechanism of interaction between FGF-2 and heparin, the association structures between human FGF-2 and different kinds of regioselectively desulfated heparins were observed by small angle X-ray scattering. In the FGF-2-native heparin complex, the global FGF-2 molecules appeared to attach along heparin chain as strained unilaterally. The complexes with the 6-O-, or N-desulfated heparin seemed to have randomly associated structure as compared with above system. On the other hand, 2-O-desulfated heparin did not indicate the aggregation with FGF-2, indicating that the sulfate groups at O-2 of iduronate residues in heparin is most essential for association with FGF-2. These structural characteristics will be deeply related with signal transduction in the association with FGF-2 receptor.

Metal ion-dependent effects of clioquinol on the fibril growth of an amyloid {beta} peptide.

Although metal ions such as Cu(2+), Zn(2+), and Fe(3+) are implicated to play a key role in Alzheimer disease, their role is rather complex, and comprehensive understanding is not yet obtained. We show that Cu(2+) and Zn(2+) but not Fe(3+) renders the amyloid beta peptide, Abeta(1-40), nonfibrillogenic in nature. However, preformed fibrils of Abeta(1-40) were stable when treated with these metal ions. Consequently, fibril growth of Abeta(1-40) could be switched on/off by switching the molecule between its apo- and holo-forms. Clioquinol, a potential drug for Alzheimer disease, induced resumption of the Cu(2+)-suppressed but not the Zn(2+)-suppressed fibril growth of Abeta(1-40). The observed synergistic effect of clioquinol and Zn(2+) suggests that Zn(2+)-clioquinol complex effectively retards fibril growth. Thus, clioquinol has dual effects; although it disaggregates the metal ion-induced aggregates of Abeta(1-40) through metal chelation, it further retards the fibril growth along with Zn(2+). These results indicate the mechanism of metal ions in suppressing Abeta amyloid formation, as well as providing information toward the use of metal ion chelators, particularly clioquinol, as potential drugs for Alzheimer disease.

Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy.

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb5 was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb5 molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb5 molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.

Critical balance of electrostatic and hydrophobic interactions is required for beta 2-microglobulin amyloid fibril growth and stability.

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.

Direct observation of Abeta amyloid fibril growth and inhibition.

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.