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Study is presented to demonstrate that monomeric A(beta) undergoes an immediate conversion to a predominant beta-structured conformation in 2 mM sodium dodecylsulfate (SDS) which does not proceed to amyloid fibrils. It is seen that endogenous oligomeric A(beta) aggregates are stabilized by undefined process.

Series of evidence which provide insight into significant application of soluble amyloid-[beta] (A[beta]) oligomers as well as fibrils in the etiology of Alzheimer's disease (AD) are discussed. The need to develop optimal mass spectrometry (MS) analysis procedures to explore and explain the hypothesis related to the stabilization of endogenous A[beta] oligomers by covalent cross-linking induced via unknown cellular agents are also discussed.

A focus is laid on characterizing the energetics and sequence of metal binding to diphtheria toxin repressor (DtxR) using Ni(II), Cd(II) and Mn(II). The results of this study extend the description of metal binding energetics in diphtheria toxin repressor (DtxR) and demonstrate a specific sequence of events leading to the activation of the aporepressor.

A study was carried out to determine the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by using the NMR spectroscopy. The structural and biophysical studies suggest that the proline-rich peptide segment of diphtheria toxin repressor functions as a switch that modulates the activation of repressor activity.

A neuropathological hallmark of Alzheimer's disease (AD) is the presence of large numbers of senile plaques in the brain. These deposits are rich in fibrils that are composed of 40- and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that soluble Aβ aggregates as well as fibrils are important in the etiology of AD. Low levels of endogenous soluble Aβ aggregates make them difficult to characterize, but several species in extracts of AD brains have been detected by gel electrophoresis in sodium dodecyl sulfate (SDS) and immunoblotting. Individual Aβ oligomers ranging in size from dimers through dodecamers of 4 kDa monomeric Aβ have been resolved in other laboratories as discrete species by size exclusion chromatography (SEC). In an effort to reconstitute soluble Aβ aggregates in vitro that resemble the endogenous soluble Aβ aggregates, we previously found that monomeric Aβ(1-42) rapidly forms soluble oligomers in the presence of dilute SDS micelles. Here we extend this work in two directions. First, we contrast the size and secondary structure of these oligomers with those of synthetic Aβ(1-42) fibrils. SEC and multiangle light scattering were used to obtain a molecular mass of 150 kDa for the isolated oligomers. The oligomers partially dissociated to monomers through nonamers when incubated with SDS, but in contrast to endogenous oligomers, we saw no evidence of these discrete species prior to SDS treatment. One hypothesis to explain this difference is that endogenous oligomers are stabilized by covalent cross-linking induced by unknown cellular agents. To explore this hypothesis, optimal mass spectrometry (MS) analysis procedures need to be developed for Aβ cross-linked in vitro. In our second series of studies, we began this process by treating monomeric and aggregated Aβ(1-42) with three cross-linking agents: transglutamitiase, glutaraldehyde, and Cu(II) with peroxide. We compared the efficiency of covalent cross-linking with these agents, the effect of cross-linking on peptide secondary structure, the stability of the cross-linked structures to thermal unfolding, and the sites of peptide cross-linking obtained from proteolysis and MS. [ABSTRACT FROM AUTHOR]

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-β (Aβ) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Aβ species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Aβ 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 Aβ, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Aβ aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Aβ aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Aβ(1—40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Aβ(1—42) undergoes an immediate conversion to a predominant β-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 Aβ aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Aβ aggregation procedures. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that fibrillar Aβ and especially soluble Aβ aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Aβ aggregates generated from monomeric Aβ in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily β-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Aβ aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Aβ aggregation have found that amphiphilic interactions and micellar-like Aβ structures may play a role. We previously described the formation of Aβ(1–40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Aβ(1–40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Aβ(1–40) was introduced. With HFIP, the change involved an increase in predominant/3-structure content and in fluorescence with thioflavin T, while with SDS, a partial α-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in β-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Aβ(1–40) monomers effectively. [ABSTRACT FROM AUTHOR]