Alternate aggregation pathways of the Alzheimer beta-amyloid peptide: Abeta association kinetics at endosomal pH

The deposition of beta-amyloid peptide (Abeta) fibrils around neurons is an invariable feature of Alzheimer's disease and there is increasing evidence that fibrillar deposits and/or prefibrillar intermediates play a central role in the observed neurodegeneration. One site of Abeta generation is the endosomes, and we have investigated the kinetics of Abeta association at endosomal pH over physiologically relevant time frames. We have identified three distinct Abeta association phases that occur at rates comparable to endosomal transit times. Rapid formation of burst phase aggregates, larger than 200nm, was observed within 15 seconds. Two slower association phases were detected by fluorescence resonance energy transfer and termed phase 1 and phase 2 aggregation reactions. At 20 microM Abeta, pH 6, the half lives of the phase 1 and phase 2 aggregation phases were 3.15 minutes and 17.66 minutes, respectively. Atomic force microscopy and dynamic light scattering studies indicate that the burst phase aggregate is large and amorphous, while phase 1 and 2 aggregates are spherical with hydrodynamic radii around 30 nm. There is an apparent equilibrium, potentially mediated through a soluble Abeta intermediate, between the large burst phase aggregates and phase 1 and 2 spherical particles. The large burst phase aggregates form quickly, however, they disappear as the equilibrium shifts toward the spherical aggregates. These aggregated species do not contain alpha-helical or beta-structure as determined by circular dichroism spectroscopy. However, after two weeks beta-structure is observed and is attributable to the insoluble portion of the sample. After two months, mature amyloid fibrils appear and the spherical aggregates are significantly diminished.     

Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association

Calmodulin (CaM)-kinase II holoenzymes composed of either alpha or beta subunits were analyzed using light scattering to determine a mechanism for self-association. Under identical reaction conditions, only alphaCaM-kinase II holoenzymes self-associated. Self-association was detected at a remarkably low enzyme concentration (0.14 microM or 7 microg/mL). Light scattering revealed two phases of self-association: a rapid rise that peaked, followed by a slower decrease that stabilized after 2-3 min. Electron microscopy identified that the rapid rise in scattering was due to the formation of loosely packed clusters of holoenzymes that undergo further association into large complexes of several microns in diameter over time. Self-association required activation by Ca(2+)/CaM and was strongly dependent on pH. Self-association was not detected at pH 7.5, however, the extent of this process increased as reaction pH decreased below 7.0. A peptide substrate (autocamtide-2) and inhibitor (AIP) designed from the autoregulatory domain of CaM-kinase II potently prevented self-association, whereas the peptide substrate syntide-2 did not. Thus, CaM-kinase II self-association is isoform specific, regulated by the conditions of activation, and is inhibited by peptides that bind to the catalytic domain likely via their autoregulatory-like sequence. A model for CaM-kinase II self-association is presented whereby catalytic domains in one holoenzyme interact with the regulatory domains in neighboring holoenzymes. These intersubunit-interholoenzyme autoinhibitory interactions could contribute to both the translocation and inactivation of CaM-kinase II previously reported in models of ischemia.     

The amyloid beta peptide (Abeta(1-40)) is thermodynamically soluble at physiological concentrations

Precipitation of the 39-43-residue amyloid beta peptide (Abeta) is a crucial factor in Alzheimer's disease (AD). In normal as well as in AD-afflicted brain, the Abeta concentration is estimated to be a few nanomolar. Here we show that Abeta(1-40) precipitates in vitro only if the dissolved concentration is >14 microM. Using fluorescence correlation spectroscopy, we further show that the precipitation is complete in 1 day, after which the size distribution of Abeta monomer/oligomers in the solution phase becomes stationary in time and independent of the starting Abeta concentration. Mass spectra confirm that both the solution phase and the coexisting precipitate contain chemically identical Abeta molecules. Incubation at 68 degrees C for 1 h reduces the solubility by <12%. Together, these results show that the thermodynamic saturation concentration (C(sat)) of Abeta(1-40) in phosphate-buffered saline (PBS) at pH 7.4 has a well-defined lower limit of 15.5 +/- 1 microM. Divalent metal ions (believed to play a role in AD) at near-saturation concentrations in PBS reduce C(sat) only marginally (2 mM Mg(2+) by 6%, 2.5 microM Ca(2+) by 7%, and 4 microM Zn(2+) by 11%). Given that no precipitation is possible at concentrations below C(sat), we infer that coprecipitant(s), and not properties of Abeta(1-40) alone, are key factors in the in vivo aggregation of Abeta.     

Correlation of beta-amyloid aggregate size and hydrophobicity with decreased bilayer fluidity of model membranes

beta-amyloid peptide (Abeta) is the primary constituent of senile plaques, a defining feature of Alzheimer's disease. Aggregated Abeta is toxic to neurons, but the mechanism of toxicity remains unproven. One proposal is that Abeta toxicity results from relatively nonspecific Abeta-membrane interactions. We hypothesized that Abeta perturbs membrane structure as a function of the aggregation state of Abeta. Toward exploring this hypothesis, Abeta aggregate size and hydrophobicity were characterized using dynamic and static light scattering and 1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid (bis-ANS) fluorescence. The effect of Abeta aggregation state on the membrane fluidity of unilamellar liposomes was assessed by monitoring the anisotropy of the membrane-embedded fluorescent dye, 1,6-diphenyl-1,3,5-hexatriene (DPH). Unaggregated Abeta at pH 7 did not bind bis-ANS and had little to no effect on membrane fluidity. More significantly, Abeta aggregated at pH 6 or 7 decreased membrane fluidity in a time- and dose-dependent manner. Aggregation rate and surface hydrophobicity were considerably greater for Abeta aggregated at pH 6 than at neutral pH and were strongly correlated with the extent of decrease in membrane fluidity. Prolonged (7 days) Abeta aggregation resulted in a return to near-baseline levels in both bis-ANS fluorescence and DPH anisotropy at pH 7 but not at pH 6. The addition of gangliosides to the liposomes significantly increased the DPH anisotropy response. Hence, self-association of Abeta monomers into aggregates exposes hydrophobic sites and induces a decrease in membrane fluidity. Abeta aggregate-induced changes in membrane physical properties may have deleterious consequences on cellular functioning.     

Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.     

Selective destabilization of soluble amyloid beta oligomers by divalent metal ions

Aggregation of the amyloid beta (Abeta) peptide yields both fibrillar precipitates and soluble oligomers, and is associated with Alzheimer's disease (AD). In vitro, Cu(2+) and Zn(2+) strongly bind Abeta and promote its precipitation. However, less is known about their interactions with the soluble oligomers, which are thought to be the major toxic species responsible for AD. Using fluorescence correlation spectroscopy to resolve the various soluble species of Abeta, we show that low concentrations of Cu(2+) (1 microM) and Zn(2+) (4 microM) selectively eliminate the oligomeric population (within approximately 2h), while Mg(2+) displays a similar effect at a higher concentration (60 microM). This uncovers a new aspect of Abeta-metal ion interactions, as precipitation is not substantially altered at these low metal ion concentrations. Our results suggest that physiological concentrations of Cu(2+) and Zn(2+) can critically alter the stability of the toxic Abeta oligomers and can potentially control the course of neurodegeneration.     

Amyloid-beta(1-42) rapidly forms protofibrils and oligomers by distinct pathways in low concentrations of sodium dodecylsulfate

Alzheimer's disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-beta (Abeta) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Abeta species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Abeta are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Abeta, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Abeta aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Abeta aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Abeta(1-40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Abeta(1-42) undergoes an immediate conversion to a predominant beta-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8-14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30-60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Abeta aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Abeta aggregation procedures.     

Cu(II) binding to monomeric, oligomeric, and fibrillar forms of the Alzheimer's disease amyloid-beta peptide

Copper has been proposed to play a role in Alzheimer's disease through interactions with the amyoid-beta (Abeta) peptide. The coordination environment of bound copper as a function of Cu:Abeta stoichiometry and Abeta oligomerization state are particularly contentious. Using low-temperature electron paramagnetic resonance (EPR) spectroscopy, we spectroscopically distinguish two Cu(II) binding sites on both soluble and fibrillar Abeta (for site 1, A parallel = 168 +/- 1 G and g parallel = 2.268; for site 2, A parallel = 157 +/- 2 G and g parallel = 2.303). When fibrils that have been incubated with more than 1 equiv of Cu(II) are washed, the second Cu(II) ion is removed, indicating that it is only weakly bound to the fibrils. No change in the Cu(II) coordination environment is detected by EPR spectroscopy of Cu(II) with Abeta (1:1 ratio) collected as a function of Abeta fibrillization time, which indicates that the Cu(II) environment is independent of Abeta oligomeric state. The initial Cu(II)-Abeta complexes go on to form Cu(II)-containing Abeta fibrils. Transmission electron microscopy images of Abeta fibrils before and after Cu(II) addition are the same, showing that once incorporated, Cu(II) does not affect fibrillar structure; however, the presence of Cu(II) appears to induce fibril-fibril association. On the basis of our results, we propose a model for Cu(II) binding to Abeta during fibrillization that is independent of peptide oligomeric state.     

Exaggerated polymerisation of beta-amyloid 40 stimulated by plasma lipoproteins results in fibrillar Abeta preparations that are ineffective in promoting ADP-induced platelet aggregation

The cytotoxic beta-amyloid peptide (Abeta) of Alzheimer's disease (AD) occurs in both plasma and platelets and may modulate platelet function. Its biological activity may relate to its fibril content and factors that promote Abeta fibrillogenesis, e.g., plasma lipoproteins could, therefore, have implications for Abeta action. We undertook a study in which structure-activity relationships were considered with respect to the actions of Abeta(1-40) on platelet function. Thus, the influence of soluble Abeta and various fibrillar Abeta preparations (0.1-10 microM) on platelet aggregation and endogenous 5-hydroxytryptamine (5-HT) efflux was investigated. Soluble Abeta(1-40) only enhanced platelet aggregation (+30%, P<0.05) and 5-HT release (+28%) stimulated by ADP (1 microM) at the highest concentration tested (10 microM). By contrast, fibrillar Abeta(1-40) at 1, 5 and 10 microM potentiated aggregation by 17.4%, 68.8% (P<0.05) and 99.5% (P<0.0001), respectively, and 5-HT efflux by 17.4%, 65% and 208% (P<0.001). Abeta(1-40) fibrils generated in the presence of native and oxidised very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) yielded platelet responses that did not differ from those seen with the lipoproteins alone. These responses were markedly lower than those obtained with homogeneous Abeta fibrils. Our data indicate that homogeneous Abeta(1-40) fibrils are more potent than soluble Abeta(1-40) in promoting platelet reactivity and that interactions with plasma lipoproteins result in the formation of Abeta fibrils that are ineffective. We suggest that lipoproteins may interfere with the recognition of Abeta by appropriate platelet receptors and/or cause Abeta to assume an "overaggregated" biologically inert state.     

Oligomerization of hydrophobin SC3 in solution: from soluble state to self-assembly

Hydrophobin SC3 is a protein with special self-association properties that differ depending on whether it is in solution, on an air/water interface or on a solid surface. Its self-association on an air/water interface and solid surface have been extensively characterized. The current study focuses on its self-association in water because this is the starting point for the other two association processes. Size-exclusion chromatography was used to fractionate soluble-state SC3. Real-time multiangular light scattering detection of the eluate indicated that SC3 mainly exists as a dimer in buffer, accompanied with a small amount of monomer, tetramer, and larger aggregates. Dimeric SC3 has very likely an elongated shape, as indicated by the hydrodynamic radius determined by using dynamic light scattering (DLS) and fluorescence anisotropy measurements on dansyl-labeled SC3. Size-exclusion chromatography experiments also indicated that the protein oligomerizes very slowly at low temperature (4 degrees C) but rather rapidly at room temperature. Ionic strength plays an important role in the oligomerization; a short-lived monomeric SC3 species could be observed in pure water. Oligomerization was not affected by low pH but was accelerated by high pH. Fluorescence resonance energy transfer showed that dissociation occurred when the protein concentration was lowered; a large population of oligomers, presumably dimers, dissociate when the protein concentration is <4.5 microg/mL. This value is similar to the critical concentration for SC3 self-assembly. Therefore, dimeric SC3 is indicated to be the building block for both aggregation in solution and self-assembly at hydrophobic/hydrophilic interfaces.     

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. I. Analysis of apparent weight-average molecular weight data for the apoenzyme in terms of models

The self-association of D-amino acid oxidase apoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was studied by low-angle laser light scattering. The concentration (c) dependence of the apparent weight-average molecular weight (Mwapp) was determined over a wide concentration range of 0.04 to 6.1 mg/ml. The extrapolated Mwapp value, to zero enzyme concentration, corresponded to the Mr value of the monomer. The self-association mode of the apoenzyme was systematically explored with nonlinear least-squares analysis of the Mwapp versus c data. The simplest model that fitted the data well was a model of isodesmic indefinite self-association of the monomer with the isodesmic association constant of 0.467 +/- 0.034 liter/g. The monomer-dimer model proposed previously, but only in a low enzyme concentration range of less than 0.9 mg/ml at 5-20 degrees C (Henn, S. W., and Ackers, G. K. (1969) Biochemistry 8, 3829-3838), did not fit the Mwapp versus c data either in the limited low concentration range or in the whole concentration range examined at 25 degrees C. To test the validity of the chosen model, the observed sedimentation boundary profiles were compared with the idealized boundary profiles calculated for the better-fit models. The profile calculated with the model of the isodesmic indefinite self-association mechanism was qualitatively consistent with the observed ones. The utility of the nonlinear least-squares procedure for analyzing self-associating systems was demonstrated.     

HIV Rev self-assembly is linked to a molten-globule to compact structural transition

By regulating the differential expression of proviral pre mRNA in the host cell, Rev plays a crucial role in the HIV-1 life cycle. The capacity of Rev to function is intimately linked to its ability to self-associate. Nevertheless, little is known about the exact role of self-association in the molecular mechanism defining its biological activity. A prerequisite knowledge is a definition of the molecular events undertaken by Rev during the process of self-assembly. Thus, this study was initiated to monitor the structure of Rev as a function of protein concentration. Rev undergoes a structural transition as a consequence of self-assembly. This structural transition was monitored by three spectroscopic methods. The accessibility of the single tryptophan in Rev monomer to acrylamide quenching increases with decreasing protein concentration. At very low concentration of Rev, the tryptophan accessibility is close to that of an unfolded Rev. As evaluated by circular dichroism, the secondary structure of Rev is protein concentration dependent as evidenced by an increase in the magnitude of ellipticity with increasing protein concentration. Further, results from ANS binding studies indicate that the ANS binding sites in Rev experience an apparent increase in hydrophobicity as the Rev concentration was increased. These concentration dependent changes seem to reach a maximum above 5 microM Rev monomer concentration. In order to define the mode of Rev self-association sedimentation velocity and equilibrium experiments were conducted. There are evidently two consecutive progressive association processes. At protein concentrations below 0.5 mg/ml, the data from sedimentation studies can be fitted to a single isodesmic model. Simulation of velocity sedimentation profile indicates that free Rev monomer that has not entered into the association processes can best be described to exhibit a value of S(20,w) that is substantially smaller than 1.4 S, a value needed to fit the rest of the data. The latter value is consistent for a Rev monomer with the expected molecules weight and if it were to assume a compact globular shape. These spectroscopic and hydrodynamic results imply that monomeric Rev is in a molten globule state, which becomes more compact upon self-association.     

Mg(2+)-Linked Self-Assembly of FtsZ in the Presence of GTP or a GTP Analogue Involves the Concerted Formation of a Narrow Size Distribution of Oligomeric Species

The assembly of the bacterial cell division FtsZ protein in the presence of constantly replenished GTP was studied as a function of Mg(2+) concentration (at neutral pH and 0.5 M potassium) under steady-state conditions by sedimentation velocity, concentration-gradient light scattering, fluorescence correlation spectroscopy, and dynamic light scattering. Sedimentation velocity measurements confirmed previous results indicating cooperative appearance of a narrow size distribution of finite oligomers with increasing protein concentration. The concentration dependence of light scattering and diffusion coefficients independently verified the cooperative appearance of a narrow distribution of high molecular weight oligomers, and in addition provided a measurement of the average size of these species, which corresponds to 100 ± 20 FtsZ protomers at millimolar Mg(2+) concentration. Parallel experiments on solutions containing guanosine-5'-[(α,β)-methyleno]triphosphate, sodium salt (GMPCPP), a slowly hydrolyzable analogue of GTP, in place of GTP, likewise indicated the concerted formation of a narrow size distribution of fibrillar oligomers with a larger average mass (corresponding to 160 ± 20 FtsZ monomers). The closely similar behavior of FtsZ in the presence of both GTP and GMPCPP suggests that the observations reflect equilibrium rather than nonequilibrium steady-state properties of both solutions and exhibit parallel manifestations of a common association scheme.     

Rapid quantitative characterization of protein interactions by composition gradient static light scattering

Two new applications of the recently developed technique of composition gradient static light scattering (CG-SLS) are presented. 1), The method is demonstrated to be capable of detecting and quantitatively characterizing reversible association of chymotrypsin and bovine pancreatic trypsin inhibitor in a solution mixture and simultaneously occurring reversible self-association of chymotrypsin at low pH in the same mixture. The values of equilibrium constants for both self- and heteroassociation may be determined with reasonable precision from the analysis of data obtained from a single experiment requiring <15 min and <1 mg of each protein. 2), Analysis of the results of a single CG-SLS experiment carried out on Ftsz, a protein that self-associates to form linear oligomers of indefinite size in the presence of guanosine diphosphate, yields the dependence of the equilibrium constant for monomer addition upon oligomer size.     

Opposing effects of inositol hexakisphosphate on rod arrestin and arrestin2 self-association

The robust cooperative formation of rod arrestin tetramers has been well-established, whereas the ability of other members of the arrestin family to self-associate remains controversial. Here, we used purified arrestins and multi-angle light scattering to quantitatively compare the propensity of the four mammalian arrestin subtypes to self-associate. Both non-visual and cone arrestins only form oligomers at very high non-physiological concentrations. However, inositol hexakisphosphate (IP6), a fairly abundant form of inositol in the cytoplasm, greatly facilitates self-association of arrestin2. Arrestin2 self-association equilibrium constants in the presence of 100 microM IP6 suggest that an appreciable proportion could exist in an oligomeric state but only in intracellular compartments where its concentration is 5-10-fold higher than average. In contrast to arrestin2, IP6 inhibits self-association of rod arrestin, indicating that the structure of these two tetramers in solution is likely different.     

New insights into the mechanism of Alzheimer amyloid-beta fibrillogenesis inhibition by N-methylated peptides

Alzheimer's disease is a debilitating neurodegenerative disorder associated with the abnormal self-assembly of amyloid-beta (Abeta) peptides into fibrillar species. N-methylated peptides homologous to the central hydrophobic core of the Abeta peptide are potent inhibitors of this aggregation process. In this work, we use fully atomistic molecular dynamics simulations to study the interactions of the N-methylated peptide inhibitor Abeta16-20m (Ac-Lys(16)-(Me)Leu(17)-Val(18)-(Me)Phe(19)-Phe(20)-NH(2)) with a model protofilament consisting of Alzheimer Abeta16-22 peptides. Our simulations indicate that the inhibitor peptide can bind to the protofilament at four different sites: 1), at the edge of the protofilament; 2), on the exposed face of a protofilament layer; 3), between the protofilament layers; and 4), between the protofilament strands. The different binding scenarios suggest several mechanisms of fibrillogenesis inhibition: 1), fibril inhibition of longitudinal growth (in the direction of monomer deposition); 2), fibril inhibition of lateral growth (in the direction of protofilament assembly); and 3), fibril disassembly by strand removal and perturbation of the periodicity of the protofilament (disruption of fibril morphology). Our simulations suggest that the Abeta16-20m inhibitor can act on both prefibrillar species and mature fibers and that the specific mechanism of inhibition may depend on the structural nature of the Abeta aggregate. Disassembly of the fibril can be explained by a mechanism through which the inhibitor peptides bind to disaggregated or otherwise free Abeta16-22 peptides in solution, leading to a shift in the equilibrium from a fibrillar state to one dominated by inhibitor-bound Abeta16-22 peptides.     

Conformational dynamics of amyloid beta-protein assembly probed using intrinsic fluorescence

Formation of toxic oligomeric and fibrillar structures by the amyloid beta-protein (Abeta) is linked to Alzheimer's disease (AD). To facilitate the targeting and design of assembly inhibitors, intrinsic fluorescence was used to probe assembly-dependent changes in Abeta conformation. To do so, Tyr was substituted in Abeta40 or Abeta42 at position 1, 10 (wild type), 20, 30, 40, or 42. Fluorescence then was monitored periodically during peptide monomer folding and assembly. Electron microscopy revealed that all peptides assembled readily into amyloid fibrils. Conformational differences between Abeta40 and Abeta42 were observed in the central hydrophobic cluster (CHC) region, Leu17-Ala21. Tyr20 was partially quenched in unassembled Abeta40 but displayed a significant and rapid increase in intensity coincident with the maturation of an oligomeric, alpha-helix-containing intermediate into amyloid fibrils. This process was not observed during Abeta42 assembly, during which small decreases in fluorescence intensity were observed in the CHC. These data suggest that the structure of the CHC in Abeta42 is relatively constant within unassembled peptide and during the self-association process. Solvent accessibility of the Tyr ring was studied using a mixed solvent (dimethyl sulfoxide/water) system. [Tyr40]Abeta40, [Tyr30]Abeta42, and [Tyr42]Abeta42 all were relatively shielded from solvent. Analysis of the assembly dependence of the site-specific intrinsic fluorescence data suggests that the CHC is particularly important in controlling Abeta40 assembly, whereas the C-terminus plays the more significant role in Abeta42 assembly.     

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. II. Stoichiometry of holoenzyme association and energetics of subunit association

The self-association pattern of D-amino acid oxidase holoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was examined by the low-angle laser light-scattering method. As to the results of nonlinear least-squares analysis of the apparent weight-average molecular weight (Mwapp) versus protein concentration (c) data, the following three models fitted equally well the data over the concentration range of 0.03-11.4 mg/ml: 1) the model of isodesmic indefinite self-association of the monomer where the dimerization constant differs from the isodesmic association constant, 2) the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, and 3) the model which involves the trimerization of the monomer and isodesmic indefinite self-association of the trimer. In a more limited concentration range (0.3-11.4 mg/ml), a model of isodesmic indefinite self-association of the stable dimer where the dimer does not dissociate into the monomers cannot be excluded from the above three models. Measurements with the concentration range lowered to 0.03 mg/ml enabled us to exclude unequivocally the model involving such a stable dimer and to extrapolate the Mwapp data to the Mr of the monomer at infinite dilution as in the case of the apoenzyme. The observed sedimentation boundary profiles were qualitatively consistent with the idealized boundary profiles calculated with the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, so this model is the most probable of the models examined. These results provide the first evidence that the association mode of the holoenzyme is different from that of the apoenzyme, i.e. isodesmic indefinite self-association of the monomer (Tojo, H., Horiike, K., Shiga, K., Nishina, Y., Watari, H., and Yamano, T. (1985) J. Biol. Chem. 260, 12607-12614). The overall linkage scheme, between binding of coenzyme FAD and subunit association, was considered, and the overall free energy change in each process in the scheme was calculated. The total stabilization energies of the intersubunit interaction in the holoenzyme relative to the apoenzyme were found to be -2.2 kcal/mol at the dimerization step and -0.5 kcal/mol at the step of the addition of the dimer to any 2i-mer (i = 1,2, ...).