Single-molecule imaging reveals aß42:aß40 ratio-dependent oligomer growth on neuronal processes.

Soluble oligomers of the amyloid-ß peptide have been implicated as proximal neurotoxins in Alzheimer's disease. However, the identity of the neurotoxic aggregate(s) and the mechanisms by which these species induce neuronal dysfunction remain uncertain. Physiologically relevant experimentation is hindered by the low endogenous concentrations of the peptide, the metastability of Aß oligomers, and the wide range of observed interactions between Aß and biological membranes. Single-molecule microscopy represents one avenue for overcoming these challenges. Using this technique, we find that Aß binds to primary rat hippocampal neurons at physiological concentrations. Although amyloid-ß(1-40) as well as amyloid-ß(1-42) initially form larger oligomers on neurites than on glass slides, a 1:1 mix of the two peptides result in smaller neurite-bound oligomers than those detected on-slide or for either peptide alone. With 1 nM peptide in solution, Aß40 oligomers do not grow over the course of 48 h, Aß42 oligomers grow slightly, and oligomers of a 1:1 mix grow substantially. Evidently, small Aß oligomers are capable of binding to neurons at physiological concentrations and grow at rates dependent on local Aß42:Aß40 ratios. These results are intriguing in light of the increased Aß42:Aß40 ratios shown to correlate with familial Alzheimer's disease mutations.

ß-Amyloid (1-40) peptide interactions with supported phospholipid membranes: a single-molecule study.

Recent evidence supports the hypothesis that the oligomers formed by the ß-amyloid peptide early in its aggregation process are neurotoxic and may feature in Alzheimer's disease. Although the mechanism underlying this neurotoxicity remains unclear, interactions of these oligomers with neuronal membranes are believed to be involved. Identifying the neurotoxic species is challenging because ß-amyloid peptides form oligomers at very low physiological concentrations (nM), and these oligomers are highly heterogeneous and metastable. Here, we report the use of single-molecule imaging techniques to study the interactions between ß-amyloid (1-40) peptides and supported synthetic model anionic lipid membranes. The evolution of the ß-amyloid species on the membranes was monitored for up to several days, and the results indicate an initial tight, uniform, binding of ß-amyloid (1-40) peptides to the lipid membranes, followed by oligomer formation in the membrane. At these low concentrations, the behavior at early times during the formation of small oligomers is interpreted qualitatively in terms of the two-state model proposed by H. W. Huang for the interaction between amphipathic peptides and membranes. However, the rate of oligomer formation in the membrane and their size are highly dependent on the concentrations of ß-amyloid (1-40) peptides in aqueous solution, suggesting two different pathways of oligomer formation, which lead to drastically different species in the membrane and a departure from the two-state model as the concentration increases.

Direct observation of single amyloid-ß(1-40) oligomers on live cells: binding and growth at physiological concentrations.

Understanding how amyloid-ß peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric Aß interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high Aß concentrations required for most biochemical analyses, the metastable nature of Aß aggregates, and the complex variety of Aß species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small Aß(1-40) oligomers bind to living neuroblastoma cells at physiological Aß concentrations. Single particle fluorescence intensity measurements indicate that cell-bound Aß species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound Aß oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary Aß species that interact with neurons at physiological concentrations.

Simultaneous single-molecule fluorescence and conductivity studies reveal distinct classes of Abeta species on lipid bilayers.

The extracellular senile plaques prevalent in brain tissue in Alzheimer's disease (AD) are composed of amyloid fibrils formed by the Abeta peptide. These fibrils have been traditionally believed to be featured in neurotoxicity; however, numerous recent studies provide evidence that cytotoxicity in AD may be associated with low-molecular weight oligomers of Abeta that associate with neuronal membranes and may lead to membrane permeabilization and disruption of the ion balance in the cell. The underlying mechanism leading to disruption of the membrane is the subject of many recent studies. Here we report the application of single-molecule optical detection, using fluorescently labeled human Alphabeta40, combined with membrane conductivity measurements, to monitor the interaction of single-oligomeric peptide structures with model planar black lipid membranes (BLMs). In a qualitative study, we show that the binding of Alphabeta to the membrane can be described by three distinctly different behaviors, depending on the Alphabeta monomer concentration. For concentrations much below 10 nM, there is uniform binding of monomers over the surface of the membrane with no evidence of oligomer formation or membrane permeabilization. Between 10 nM and a few hundred nanomolar, the uniform monomer binding is accompanied by the presence of peptide species ranging from dimers to small oligomers. The dimers are not found to permeabilize the membrane, but the larger oligomers lead to permeabilization with individual oligomers producing ion conductances of <10 pS/pore. At higher concentrations, perhaps beyond physiologically relevant concentrations, larger extended and dynamic structures are found with large conductances (hundreds of picosiemens), suggesting a major disruption of the membrane.

Structural plasticity of an acid-activated chaperone allows promiscuous substrate binding.

HdeA has been shown to prevent acid-induced aggregation of proteins. With a mass of only 9.7 kDa, HdeA is one of the smallest chaperones known. Unlike other molecular chaperones, which are typically complex, multimeric ATP-dependent machines, HdeA is known to undergo an acid-induced dimer to monomer transition and functions at low pH as a disordered monomer without the need for energy factors. Thus, HdeA must possess features that allow it to bind substrates and regulate substrate affinity in a small and energy-independent package. To understand better how HdeA accomplishes this, we studied the conformational changes that accompany a shift to low pH and substrate binding. We find that the acid-induced partial unfolding and monomerization that lead to HdeA activation occur very rapidly (k >3.5 s(-1)). Activation exposes the hydrophobic dimer interface, which we found to be critical for substrate binding. We show by intramolecular FRET that the partially unfolded character of active HdeA allows the chaperone to adopt different conformations as required for the recognition and high-affinity binding of different substrate proteins. These efficient adaptations help to explain how a very small protein is rapidly activated and can bind a broad range of substrate proteins in a purely pH-regulated manner.

Amyloid-beta membrane binding and permeabilization are distinct processes influenced separately by membrane charge and fluidity.

The 40 and 42 residue amyloid-beta (Abeta) peptides are major components of the proteinaceous plaques prevalent in the Alzheimer's disease-afflicted brain and have been shown to have an important role in instigating neuronal degeneration. Whereas it was previously thought that Abeta becomes cytotoxic upon forming large fibrillar aggregates, recent studies suggest that soluble intermediate-sized oligomeric species cause cell death through membrane permeabilization. The present study examines the interactions between Abeta40 and lipid membranes using liposomes as a model system to determine how changes in membrane composition influence the conversion of Abeta into these toxic species. Abeta40 membrane binding was monitored using fluorescence-based assays with a tryptophan-substituted peptide (Abeta40 [Y10W]). We extend previous observations that Abeta40 interacts preferentially with negatively charged membranes, and show that binding of nonfibrillar, low molecular mass oligomers of Abeta40 to anionic, but not neutral, membranes involves insertion of the peptide into the bilayer, as well as sequential conformational changes corresponding to the degree of oligomerization induced. Significantly, while anionic membranes in the gel, liquid crystalline, and liquid ordered phases induce these conformational changes equally, membrane permeabilization is reduced dramatically as the fluidity of the membrane is decreased. These findings demonstrate that binding alone is not sufficient for membrane permeabilization, and that the latter is also highly dependent on the fluidity and phase of the membrane. We conclude that binding and pore formation are two distinct steps. The differences in Abeta behavior induced by membrane composition may have significant implications on the development and progression of AD as neuronal membrane composition is altered with age.

Mapping tissue-specific genes correlated with age-dependent changes in protein stability and function.

Biophysical measurements indicative of protein stability and function were performed on crude extracts from liver, muscle, and lens of a genetically heterogeneous mouse population. Genetic information was used to search for quantitative trait loci (QTL) that influenced the biophysical traits, with emphasis on phenotypes that previously have been shown to be altered in aged animals. Spectroscopic and enzymatic assays of crude liver and muscle tissue extracts from approximately 600 18-month-old mice, the progeny of (BALB/cJxC57BL/6J)F1 females and (C3H/HeJxDBA/2J)F1 males, were used to measure the susceptibility of a ubiquitous glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to thermal denaturation. The rate constant for thermal inactivation of GAPDH correlated with markers on chromosome 5 (D5Mit79 and D5Mit251) for muscle lysates and chromosome 15 (D15Mit63 and D15Mit100) for liver tissue. The degree of variability of inactivation rate constants, a measure of the heterogeneity of muscle GAPDH in tissue extracts, was also associated with markers on chromosome 5 (D5Mit79 and D5Mit205). In addition, spectroscopic characteristics of extracted eye lens proteins were evaluated for their susceptibility to photooxidative stress. Absorbance and fluorescence emission characteristics of the lens proteins were mapped to QTL on chromosomes 5 and 15 (D5Mit25 and D15Mit171) while the degree of heterogeneity in photochemical oxidation kinetics was associated with a marker on the chromosome 8 (D8Mit42). Recent work has shown that GAPDH possesses a number of non-glycolytic functions including DNA/RNA binding and regulation of protein expression. Tissue specific differences in GAPDH stability may have significant consequences to these alternate functions during aging.

The triplet-state lifetime of indole in aqueous and viscous environments: significance to the interpretation of room temperature phosphorescence in proteins.

The interpretation of room temperature phosphorescence studies of proteins requires an understanding of the mechanisms governing the tryptophan triplet-state lifetimes of residues fully exposed to solvent and those deeply buried in the hydrophobic core of proteins. Since solvents exposed tryptophans are expected to behave similarly to indole free in solution, it is important to have an accurate measure of the triplet state lifetime of indole in aqueous solution. Using photon counting techniques and low optical fluence (J/cm(2)), we observed the triplet-state lifetime of aqueous, deoxygenated indole and several indole derivatives to be approximately 40 micros, closely matching the previous reports by Bent and Hayon based on flash photolysis (12 micros; Bent, D. V.; Hayon, E. J. Am. Chem. Soc. 1975, 97, 2612-2619) but much shorter than the 1.2 ms lifetime observed more recently (Strambini, G. B.; Gonnelli, M. J. Am. Chem. Soc. 1995, 117, 7646-7651). However, we have now been able to reproduce the long lifetime reported by the latter workers for aqueous indole solutions and show that it likely arises from geminate recombination of the indole radical cation and solvated electron, a conclusion based on studies of the indole radical cation in water (Bent and Hayon, 1975). The evidence for this comes from a fast rise in the phosphorescence emission and measurements of a corresponding enhanced quantum yield in unbuffered solutions. This species can be readily quenched, and the corresponding fast rise disappears, leaving a monoexponential 40 micros decay, which we argue is the true indole triplet lifetime. The work is put in the context of room temperature phosphorescence studies of proteins.

The protein kinase/endoribonuclease IRE1alpha that signals the unfolded protein response has a luminal N-terminal ligand-independent dimerization domain.

In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), cells activate an intracellular signal transduction pathway called the unfolded protein response (UPR). IRE and PERK are the two type-I ER transmembrane protein kinase receptors that signal the UPR. The N-terminal luminal domains (NLDs) of IRE1 and PERK sense ER stress conditions by a common mechanism and transmit the signal to regulate the cytoplasmic domains of these receptors. To provide an experimental system amenable to detailed biochemical and structural analysis to elucidate the mechanism of ER-transmembrane signaling mechanism mediated by the NLD, we overexpressed the soluble luminal domain of human IRE1alpha in COS-1 cells by transient DNA transfection. Here we report the expression, purification, and characterization of the soluble NLD. The biological function of the NLD was confirmed by its ability to associate with itself and to interact with both the membrane-bound full-length IRE1alpha receptor and the ER chaperone BiP. Functional and spectral studies suggested that the highly conserved N-linked glycosylation site is not required for proper protein folding and self-association. Interestingly, we demonstrated that the NLD forms stable dimers linked by intermolecular disulfide bridges. Our data support that the luminal domain represents a novel ligand-independent dimerization domain.