Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces.

In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS). The data were analyzed to obtain unfolding rates and Gibbs free energies for unfolding of adsorbed proteins. The melting temperatures of the proteins were lowered, but by different amounts, on the different surfaces. In addition, the structures of the proteins on the chromatographic surfaces were similar to the partially unfolded structures produced in the absence of a surface by temperature as well as by chemical denaturants. Finally, it was found that patterns of residue exposure to solvent on different surfaces at different temperatures can be largely superimposed. These findings suggest that protein unfolding on various HIC surfaces might be quantitatively related to protein unfolding in solution and that details of surface unfolding behavior might be generalized.

Unfolding of a model protein on ion exchange and mixed mode chromatography surfaces.

Recent studies with proteins indicate that conformational changes and aggregation can occur during ion exchange chromatography (IEC). Such behavior is not usually expected, but could lead to decreased yield and product degradation from both IEC and multi mode chromatography (MMC) that has ligands of both hydrophobic and charged functionalities. In this study, we used hydrogen exchange mass spectrometry to investigate unfolding of the model protein BSA on IEC and MMC surfaces under different solution conditions at 25°C. Increased solvent exposure, indicating greater unfolding relative to that in solution, was found for protein adsorbed on cationic IEC and MMC surfaces in the pH range of 3.0 to 4.5, where BSA has decreased stability in solution. There was no effect of anionic surfaces at pH values in the range from 6.0 to 9.0. Differences of solvent exposure of whole molecules when adsorbed and in solution suggest that adsorbed BSA unfolds at lower pH values and may show aggregation, depending upon pH and the surface type. Measurements on digested peptides showed that classifications of stability can be made for various regions; these are generally retained as pH is changed. When salt was added to MMC systems, where electrostatic interactions would be minimized, less solvent exposure was seen, implying that it is the cationic moieties, rather than the hydrophobic ligands, which cause greater surface unfolding at low salt concentrations. These results suggest that proteins of lower stability may exhibit unfolding and aggregation during IEC and MMC separations, as they can with hydrophobic interaction chromatography.

Conformational stability as a design target to control protein aggregation.

Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation. A computational design approach was tested using human γD crystallin (γD-crys) as a model multi-domain protein. Two mutational strategies were tested for their ability to reduce/increase aggregation rates by increasing/decreasing ΔGunf: stabilizing the less stable domain and stabilizing the domain-domain interface. The computational protein design algorithm, RosettaDesign, was implemented to identify point variants. The results showed that although the predicted free energies were only weakly correlated with the experimental ΔGunf values, increased/decreased aggregation rates for γD-crys correlated reasonably well with decreases/increases in experimental ΔGunf, illustrating improved conformational stability as a possible design target to mitigate aggregation. However, the results also illustrate that conformational stability is not the sole design factor controlling aggregation rates of natively folded proteins.

Cis-suppression to arrest protein aggregation in mammalian cells.

Protein misfolding and aggregation are implicated in numerous human diseases and significantly lower production yield of proteins expressed in mammalian cells. Despite the importance of understanding and suppressing protein aggregation in mammalian cells, a protein design and selection strategy to modulate protein misfolding/aggregation in mammalian cells has not yet been reported. In this work, we address the particular challenge presented by mutation-induced protein aggregation in mammalian cells. We hypothesize that an additional mutation(s) can be introduced in an aggregation-prone protein variant, spatially near the original mutation, to suppress misfolding and aggregation (cis-suppression). As a model protein, we chose human copper, zinc superoxide dismutase mutant (SOD1(A4V) ) containing an alanine to valine mutation at residue 4, associated with the familial form of amyotrophic lateral sclerosis. We used the program RosettaDesign to identify Phe20 in SOD1(A4V) as a key residue responsible for SOD1(A4V) conformational destabilization. This information was used to rationally develop a pool of candidate mutations at the Phe20 site. After two rounds of mammalian-cell based screening of the variants, three novel SOD1(A4V) variants with a significantly reduced aggregation propensity inside cells were selected. The enhanced stability and reduced aggregation propensity of the three novel SOD1(A4V) variants were verified using cell fractionation and in vitro stability assays.

Reduction of the C191-C220 disulfide of α-chymotrypsinogen A reduces nucleation barriers for aggregation.

Proper disulfide formation can be essential for the conformational stability of natively folded proteins. For proteins that must unfold in order to aggregate, disruption of native disulfides may therefore promote aggregation. This study characterizes differences in the aggregation process for wild-type (WT) α-chymostrypsinogen A (aCgn) and the same molecule with one of its native disulfides (C191-C220) reduced to free thiols (aCgnSH) at acidic pH, where WT aCgn forms semi-flexible amyloid polymers. Loss of the disulfide leads to no discernable differences in folded monomer secondary or tertiary structure based on circular dichroism (CD) or intrinsic fluorescence (FL), and causes a small decrease in the free energy change upon unfolding. After unfolding-mediated aggregation, the resulting amyloid morphology and structure are similar or indistinguishable for aCgn and aCgnSH by CD, FL, ThT binding, multi-angle laser light scattering, and transmission electron microscopy. Aggregates of aCgn and aCgnSH are also able to cross-seed with monomers of the other species. However, aggregates of aCgnSH are more resistive than aCgn aggregates to urea-mediated dissociation, suggesting some degree of structural differences in the aggregated species that was not resolvable in detail without higher resolution methods. Mechanistic analyses of aggregation kinetics indicate that the initiation or nucleation of new aggregates from aCgnSH involves a mono-molecular rate limiting step, possibly the unfolding step. In contrast, that for aCgn involves an oligomeric intermediate, suggesting native disulfide linkages help to hinder non-native protein aggregation by providing conformational barriers to key nucleation event(s).

Aggregation of anti-streptavidin immunoglobulin gamma-1 involves Fab unfolding and competing growth pathways mediated by pH and salt concentration.

Changes in non-native aggregation mechanisms of an anti-streptavidin (anti-SA) IgG1 antibody were determined over a wide range of pH and [NaCl] under accelerated (high temperature) conditions, using a combination of calorimetry, chromatography, static light scattering, dye binding, and spectroscopy (fluorescence, infra-red, and circular dichroism). Aggregation rates were strongly influenced by conformational stability of at least the Fab regions, but were only weakly affected by changes in electrostatic colloidal interactions. This was in contrast to the effects of electrostatic interactions on aggregate growth, as the dominant growth mechanism shifted dramatically with pH and [NaCl]. Pre-formed aggregates also displayed a reversible cloud-point boundary that quantitatively aligned with the overall pattern of aggregation mechanisms as a function of pH and [NaCl], suggesting an underlying thermodynamic transition may dictate whether molecular aggregates will coalesce into macroscopic particles. Structural changes upon unfolding and aggregation were also sensitive to pH and [NaCl]. Interestingly, Thioflavin T binding was essentially indistinguishable for aggregates formed in different pH and [NaCl] conditions, however, the other assays indicated notable differences across different solvent conditions. This suggests that the overall degree of conformational change during aggregation can be influenced by electrostatic interactions, but suggests caution in interpreting whether available techniques detect changes that are directly relevant to the mechanism(s) of aggregate formation and growth.

Thermodynamics of amyloid dissociation provide insights into aggregate stability regimes.

Amyloid aggregates have been hypothesized as a global low free energy state for proteins at finite concentrations. Near its midpoint unfolding temperature, α-chymotrypsinogen A (aCgn) spontaneously forms amyloid polymers, indicating the free energy of aggregates (A) is significantly lower than that for unfolded (U) and native (N) monomers at those particular conditions. The relative thermodynamic stability of A, U, and N states was estimated semi-quantitatively as a function of temperature (T) and [urea] via a combination of calorimetry, urea-assisted unfolding and dissociation, aggregation kinetics, and changes in solvent-exposed surface area, combined with thermodynamic integration and a linear transfer free energy model. The results at first suggest that N is more thermodynamically stable than A at sufficiently low T and [urea], but this may be convoluted with kinetic effects. Interestingly, the kinetic stability of aggregates highlights that the practical measure of stability may be the free energy barrier(s) between A and U, as U serves as a key intermediate between N and A states.

Distinct aggregation mechanisms of monoclonal antibody under thermal and freeze-thaw stresses revealed by hydrogen exchange.

PURPOSE: Aggregation of monoclonal antibodies (mAbs) is a common yet poorly understood issue in therapeutic development. There remains a need for high-resolution structural information about conformational changes and intermolecular contacts during antibody aggregation. METHODS: We used hydrogen exchange mass spectrometry (HX-MS) to compare the aggregation mechanism and resultant aggregate structures of the pharmaceutical antibody Bevacizumab under freeze-thaw (F/T) and thermal stresses. RESULTS: Bevacizumab aggregation increased with number of F/T cycles and decreased with protein concentration. HX-MS showed native-like aggregates. Conversely, thermal stress triggered non-native aggregation at temperatures below melting point of the least stable CH2 domain. Under these conditions, HX was significantly enhanced in much of the Fab fragment while being decreased relative to native HX in CDRs. Analysis of intrinsic fluorescence Trp and extrinsic ANS dye binding supported structural differences between two antibody aggregates formed by F/T vs. thermal stresses. CONCLUSIONS: Reduced hydrogen exchange in three CDRs suggests these residues may form strong intermolecular contacts in the antibody aggregates; regions of enhanced HX indicate unfolding. Residue level modeling methods with varying levels of atomistic detail were unable to identify aggregation patterns predictively.

A new approach to explore the impact of freeze-thaw cycling on protein structure: hydrogen/deuterium exchange mass spectrometry (HX-MS).

PURPOSE: The impact of freeze-thaw (F/T) on structure integrity of protein therapeutics is poorly understood, partially due to lack of methods to detect protein structural perturbations during F/T processing in the frozen state. METHODS: A new approach of hydrogen/deuterium exchange was developed to separate and distinguish the specific impact of single freezing and F/T cycling on protein structure, using lactate dehydrogenase (LDH) as model system. RESULTS: In the freezing process, a fraction of LDH molecules that was inversely dependent on protein concentration was observed to partially denature its structure. Local structural perturbations were localized by peptide level HX analysis to the surface residues in segments 91-132, 170-237 and 288-331. In contrast, F/T cycling led to irreversible LDH aggregation with global structural unfolding. Residual solvent-protected structure was only detected in the aggregates for three segments, 13-31, 109-117 and 133-143, that were coincident with the consensus aggregation hotspots predicted by four different algorithms. CONCLUSIONS: Results indicate freezing preferentially disturbs local structure at the surface residues, consistent with ice-solution interface-mediated denaturation mechanism. F/T-induced aggregation begins as partial denaturation during freezing, but is accompanied by more comprehensive structural rearrangement during F/T cycling.

A safe, blood-brain barrier permeable triphenylmethane dye inhibits amyloid-ß neurotoxicity by generating nontoxic aggregates.

Growing evidence suggests that on-pathway amyloid-ß (Aß) oligomers are primary neurotoxic species and have a direct correlation with the onset of Alzheimer's disease (AD). One promising therapeutic strategy to block AD progression is to reduce the levels of these neurotoxic Aß species using small molecules. While several compounds have been shown to modulate Aß aggregation, compounds with such activity combined with safety and high blood-brain barrier (BBB) permeability have yet to be reported. Brilliant Blue G (BBG) is a close structural analogue of a U.S. Food and Drug Administration (FDA)-approved food dye and has recently garnered prominent attention as a potential drug to treat spinal cord injury due to its neuroprotective effects along with BBB permeability and high degree of safety. In this work, we demonstrate that BBG is an effective Aß aggregation modulator, which reduces Aß-associated cytotoxicity in a dose-dependent manner by promoting the formation of off-pathway, nontoxic aggregates. Comparative studies of BBG and three structural analogues, Brilliant Blue R (BBR), Brilliant Blue FCF (BBF), and Fast Green FCF (FGF), revealed that BBG is most effective, BBR is moderately effective, and BBF and FGF are least effective in modulating Aß aggregation and cytotoxicity. Therefore, the two additional methyl groups of BBG and other structural differences between the congeners are important in the interaction of BBG with Aß leading to formation of nontoxic Aß aggregates. Our findings support the hypothesis that generating nontoxic aggregates using small molecule modulators is an effective strategy for reducing Aß cytotoxicity. Furthermore, key structural features of BBG identified through structure-function studies can open new avenues into therapeutic design for combating AD.

Computational design and biophysical characterization of aggregation-resistant point mutations for γD crystallin illustrate a balance of conformational stability and intrinsic aggregation propensity.

γD crystallin is a natively monomeric eye-lens protein that is associated with hereditary juvenile cataract formation. It is an attractive model system as a multidomain Greek-key protein that aggregates through partially folded intermediates. Point mutations M69Q and S130P were used to test (1) whether the protein-design algorithm RosettaDesign would successfully predict mutants that are resistant to aggregation when combined with informatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (ΔΔG(un)) and intrinsic aggregation propensity (IAP). M69Q was predicted to have ΔΔG(un) ≫ 0, without significantly affecting IAP. S130P was predicted to have ΔΔG(un) ∼ 0 but with reduced IAP. The stability, conformation, and aggregation kinetics in acidic solution were experimentally characterized and compared for the variants and wild-type (WT) protein using circular dichroism and intrinsic fluorescence spectroscopy, calorimetric and chemical unfolding, thioflavin-T binding, chromatography, static laser light scattering, and kinetic modeling. Monomer secondary and tertiary structures of both variants were indistinguishable from WT, while ΔΔG(un) > 0 for M69Q and ΔΔG(un) < 0 for S130P. Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its IAP compared to WT and M69Q.

Protein stability and structure in HIC: hydrogen exchange experiments and COREX calculations.

Hydrogen exchange mass spectrometry (HXMS) coupled to proteolytic digestion has been used to probe the conformation of bovine ß-lactoglobulin (BLG), bovine α-lactalbumin (BLA), and human serum albumin (HSA) in solution and while adsorbed to the hydrophobic interaction chromatography media Phenyl Sepharose 6FF. All three proteins show evidence of EX1 exchange kinetics, indicating a loss of stability on the surface. HX protection patterns for all three proteins also indicate that the unfolded form is only partially solvent exposed. The hydrogen-deuterium exchange patterns of BLG and BLA on the surface suggest a structure that resembles each protein's respective solution phase molten globule state. The low stability of Domain II of HSA observed on Phenyl Sepharose 6FF also suggests a link to solution stability because Domain II is frequently cited as the least stable domain in solution unfolding pathways. COREX, an algorithm used to compute protein folding stabilities, correctly predicts solution hydrogen-deuterium exchange patterns for BLG and offers insight into its adsorbed phase stabilities but is unreliable for BLA predictions. The results of this work demonstrate a link between solution-phase local stability patterns and the nature of partially unfolded states that proteins can adopt on HIC surfaces.

Molecular level insights into thermally induced α-chymotrypsinogen A amyloid aggregation mechanism and semiflexible protofibril morphology.

Understanding nonnative protein aggregation is critical not only to a number of amyloidosis disorders but also for the development of effective and safe biopharmaceuticals. In a series of previous studies [Weiss et al. (2007) Biophys. J. 93, 4392-4403; Andrews et al. (2007) Biochemistry 46, 7558-7571; Andrews et al. (2008) Biochemistry 47, 2397-2403], α-chymotrypsinogen A (aCgn) and bovine granulocyte colony stimulating factor (bG-CSF) have been shown to exhibit the kinetic and morphological features of other nonnative aggregating proteins at low pH and ionic strength. In this study, we investigated the structural mechanism of aCgn aggregation. The resultant aCgn aggregates were found to be soluble and exhibited semiflexible filamentous aggregate morphology under transmission electron microscopy. In addition, the filamentous aggregates were demonstrated to possess amyloid characteristics by both Congo red binding and X-ray diffraction. Peptide level hydrogen exchange (HX) analysis suggested that a buried native ß-sheet comprised of three peptide segments (39-46, 51-64, and 106-114) reorganizes into the cross-ß amyloid core of aCgn aggregates and that at least ∼50% of the sequence adopts a disordered structure in the aggregates. Furthermore, the equimolar, bimodal HX labeling distribution observed for three reported peptides (65-102, 160-180, and 229-245) suggested a heterogeneous assembly of two molecular conformations in aCgn aggregates. This demonstrates that extended ß-sheet interactions typical of the amyloid are sufficiently strong that a relatively small fraction of polypeptide sequence can drive formation of filamentous aggregates even under conditions favoring colloidal stability.

Evaluation of polymer matrices for an adsorptive approach to plasma detoxification.

Acute liver failure arises when potentially toxic metabolites accumulate in the bloodstream because of a breakdown in liver function. New extracorporeal systems combining membrane and adsorbent technologies are being developed to replace critical liver detoxification functions between diagnosis and transplantation. This study addresses the adsorption of representative plasma components on four different hydrophobic, polymeric adsorbents for possible use in an extracorporeal hemodialysis device. The adsorbents considered span a range of pore sizes and include both strongly hydrophobic divinylbenzene (DVB) matrices as well as a less hydrophobic acrylate matrix. Adsorption equilibrium and rate measurements were made for these matrices using human serum albumin (HSA), polyclonal human immunoglobulin G (IgG), and bilirubin (BR), as representative plasma components. Pore size was found to contribute significantly to selectivity. Results demonstrated that strongly hydrophobic materials with pore sizes that allow free access to protein-bound BR are most effective for BR removal whether they are initially clean or pre-saturated with HSA.

The solution structure and dynamics of the DH-PH module of PDZRhoGEF in isolation and in complex with nucleotide-free RhoA.

The DH-PH domain tandems of Dbl-homology guanine nucleotide exchange factors catalyze the exchange of GTP for GDP in Rho-family GTPases, and thus initiate a wide variety of cellular signaling cascades. Although several crystal structures of complexes of DH-PH tandems with cognate, nucleotide free Rho GTPases are known, they provide limited information about the dynamics of the complex and it is not clear how accurately they represent the structures in solution. We used a complementary combination of nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and hydrogen-deuterium exchange mass spectrometry (DXMS) to study the solution structure and dynamics of the DH-PH tandem of RhoA-specific exchange factor PDZRhoGEF, both in isolation and in complex with nucleotide free RhoA. We show that in solution the DH-PH tandem behaves as a rigid entity and that the mutual disposition of the DH and PH domains remains identical within experimental error to that seen in the crystal structure of the complex, thus validating the latter as an accurate model of the complex in vivo. We also show that the nucleotide-free RhoA exhibits elevated dynamics when in complex with DH-PH, a phenomenon not observed in the crystal structure, presumably due to the restraining effects of crystal contacts. The complex is readily and rapidly dissociated in the presence of both GDP and GTP nucleotides, with no evidence of intermediate ternary complexes.

Two disaccharides and trimethylamine N-oxide affect Abeta aggregation differently, but all attenuate oligomer-induced membrane permeability.

Interaction between aggregates of amyloid beta protein (Abeta) and membranes has been hypothesized by many to be a key event in the mechanism of neurotoxicity associated with Alzheimer's disease (AD). Proposed membrane-related mechanisms of neurotoxicity include ion channel formation, membrane disruption, changes in membrane capacitance, and lipid membrane oxidation. Recently, osmolytes such as trehalose have been found to delay Abeta aggregation in vitro and reduce neurotoxicity. However, no direct measurements have separated the effects of osmolytes on Abeta aggregation versus membrane interactions. In this article, we tested the influence of trehalose, sucrose and trimethylamine-N-oxide (TMAO) on Abeta aggregation and fluorescent dye leakage induced by Abeta aggregates from liposomes. In the absence of lipid vesicles, trehalose and sucrose, but not TMAO, were found to delay Abeta aggregation. In contrast, all of the osmolytes significantly attenuated dye leakage. Dissolution of preformed Abeta aggregates was excluded as a possible mechanism of dye leakage attenuation by measurements of Congo red binding as well as hydrogen-deuterium exchange detected by mass spectrometry (HX-MS). However, the accelerated conversion of high order oligomers to fibril caused by vesicles did not take place if any of the three osmolytes presented. Instead, in the case of disaccharide, osmolytes were found to form adducts with Abeta, and change the dissociation dynamics of soluble oligomeric species. Both effects may have contributed to the observed osmolyte attenuation of dye leakage. These results suggest that disaccharides and TMAO may have very different effects on Abeta aggregation because of the different tendencies of the osmolytes to interact with the peptide backbone. However, the effects on Abeta membrane interaction may be due to much more general phenomena associated with osmolyte enhancement of Abeta oligomer stability and/or direct interaction of osmolyte with the membrane surface.

Structural understanding of stabilization patterns in engineered bispecific Ig-like antibody molecules.

Bispecific immunoglobulin-like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin-beta receptor (LTbetaR) binding Fv domain of an anti-LTbetaR/anti-TNF-related apoptosis inducing ligand receptor-2 (TRAIL-R2) bispecific immunoglobulin-like antibody. We further describe a new hierarchical structure-guided approach toward engineering of antibody-like molecules to enhance their thermal and chemical stability.

Kinetic study of beta-amyloid residue accessibility using reductive alkylation and mass spectrometry.

Beta-amyloid peptide (Abeta) is the major protein constituent found in senile plaques in Alzheimer's disease (AD). It is believed that Abeta plays a role in neurodegeneration associated with AD and that its toxicity is related to its structure or aggregation state. In this study, an approach based on chemical modification of primary amines and mass spectrometric (MS) detection was used to identify residues on Abeta peptide that were exposed or buried upon changes in peptide structure associated with aggregation. Results indicate that the N terminus was the most accessible primary amine in the fibril, followed by lysine 28, then lysine 16. A kinetic analysis of the data was then performed to quantify differences in accessibility between these modification sites. We estimated apparent equilibrium unfolding constants for each modified site of the peptide, and determined that the unfolding constant for the N terminus was approximately 100 times greater than that for K28, which was about six times greater than that for K16. Understanding Abeta peptide structure at the residue level is a first step in designing novel therapies for prevention of Abeta structural transitions and/or cell interactions associated with neurotoxicity in Alzheimer's disease.

Membrane interactions of a self-assembling model peptide that mimics the self-association, structure and toxicity of Abeta(1-40).

Beta-amyloid peptide (Abeta) is a primary protein component of senile plaques in Alzheimer's disease (AD) and plays an important, but not fully understood role in neurotoxicity. Model peptides with the demonstrated ability to mimic the structural and toxicity behavior of Abeta could provide a means to evaluate the contributions to toxicity that are common to self-associating peptides from many disease states. In this work, we have studied the peptide-membrane interactions of a model beta-sheet peptide, P(11-2) (CH(3)CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH(2)), by fluorescence, infrared spectroscopy, and hydrogen-deuterium exchange. Like Abeta(1-40), the peptide is toxic, and conditions which produce intermediate oligomers show higher toxicity against cells than either monomeric forms or higher aggregates of the peptide. Further, P(11-2) also binds to both zwitterionic (POPC) and negatively charged (POPC:POPG) liposomes, acquires a partial beta-sheet conformation in presence of lipid, and is protected against deuterium exchange in the presence of lipids. The results show that a simple rationally designed model beta-sheet peptide recapitulates many important features of Abeta peptide structure and function, reinforcing the idea that toxicity arises, at least in part, from a common mode of action on membranes that is independent of specific aspects of the amino acid sequence. Further studies of such well-behaved model peptide systems will facilitate the investigation of the general principles that govern the molecular interactions of aggregation-prone disease-associated peptides with cell and/or membrane surfaces.

Structural differences between Abeta(1-40) intermediate oligomers and fibrils elucidated by proteolytic fragmentation and hydrogen/deuterium exchange.

The aggregation of amyloid-beta protein (Abeta) in vivo is a critical pathological event in Alzheimer's disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimer's disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Abeta(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Abeta peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.