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The active sites of metalloproteins may be mimicked by designing peptides that bind to their respective metal ions. Studying the binding of protein ligands to metal ions along with the associated structural changes is important in understanding metal uptake, transport and electron transfer functions of proteins. Copper-binding metalloprotein azurin is a 128-residue electron transfer protein with a redox-active copper cofactor. Here, we report the copper-binding associated spectroscopic and structural properties of peptide loops (11 and 13 residues) from the copper-binding site of azurin. These peptides develop a β-turn upon copper-binding with a 1:1 Cu 2+ :peptide stoichiometry as seen in circular dichroism and exhibit electronic transitions centered at 340 nm and 540 nm. Further addition of copper develops a helical feature along with a shift in the absorption maxima to ~360 nm and ~580 nm at 2:1 Cu 2+ :peptide stoichiometry, indicating stoichiometric dependence of copper-binding geometry. Mass spectrometry indicates the copper-binding to cysteine, histidine and methionine in the peptide with 1:1 stoichiometry, and interestingly, dimerization through a disulfide linkage at 2:1 stoichiometry, as observed previously for denatured azurin. Fluorescence quenching studies on peptides with tryptophan further confirm the copper-binding induced changes in the two peptides are bi-phasic., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The residue lysine 28 (K28) is known to form an important salt bridge that stabilizes the Aβ amyloid structure, and acetylation of lysine 28 (K28Ac) slows the Aβ42 fibrillization rate but does not affect fibril morphology. On the other hand, acetylation of lysine 16 (K16Ac) residue greatly diminishes the fibrillization property of Aβ42 peptide and also affects its toxicity. This is due to the fact that lysine 16 acetylated amyloid beta peptide forms amorphous aggregates instead of amyloid fibrils. This is likely a result of increased hydrophobicity of the K16-A21 region due to K16 acetylation, as confirmed by molecular dynamic simulation studies. The calculated results show that the hydrophobic patches of aggregates from acetylated peptides were different when compared to wild-type (WT) peptide. K16Ac and double acetylated (KKAc) peptide aggregates show significantly higher cytotoxicity compared to the WT or K28Ac peptide aggregates alone. However, the heterogeneous mixture of WT and acetylated Aβ42 peptide aggregates exhibited higher free radical formation as well as cytotoxicity, suggesting dynamic interactions between different species could be a critical contributor to Aβ pathology.

Deposition of amyloid-β (Aβ) aggregates in the brain is a main pathological hallmark of Alzheimer's disease (AD), so inhibition of Aβ aggregation has been considered as a promising strategy for AD prevention and treatment. Black phosphorus (BP) is a 2D nanomaterial with high biocompatibility and unique biodegradability, but its potential application in biomedicine suffers from the rapid degradability and unfunctionability. To overcome the drawbacks and broaden its application, we have herein designed an Aβ inhibitor (LK7)-coupled and polyethylene glycol (PEG)-stabilized BP-based nanosystem. The PEGylated-LK7-BP nanosheets (PEG-LK7@BP) not only exhibited a good stability but also demonstrated a significantly enhanced inhibitory potency on Aβ 42 fibrillogenesis in comparison with its counterparts. This elaborately designed PEG-LK7@BP stopped the conformational transition and suppressed the fibrillization of Aβ 42 , so it could completely rescue cultured cells from the toxicity of Aβ 42 (by increasing the cell viability from 72 to 100%) at 100 μg/mL. It is considered that PEG-LK7@BP could bind Aβ species by enhanced electrostatic and hydrophobic interactions and thus efficiently alleviated Aβ-Aβ interactions. Meanwhile, the coupled LK7 on the BP surface formed a high local concentration that enhanced the affinity between the nanosystem and Aβ species. Finally, PEG could improve the stability and dispersibility of the nanoplatform to make it show an increased inhibitory effect on the amyloid formation. Hence, this work proved that PEG-LK7@BP is a promising nanosystem for the development of amyloid inhibitors fighting against AD.

Aberrant aggregation of the Aβ protein is a hallmark of Alzheimer's disease (AD), but no complete characterization of the molecular level pathogenesis has been achieved. A promising hypothesis is that dysfunction of metal ion homeostasis, and consequently, the undesired interaction of metal ions with Aβ, may be central to the development of AD. Qualitatively, most data indicate that Cu(II) induces rapid self-assembly of both Aβ40 and Aβ42 during the initial phase of the aggregation, while at longer time scales fibrillation may occur, depending on the experimental conditions. For Aβ40 and Cu(II):Aβ ≤ 1, most data imply that low concentration of Aβ40 favors nucleation and rapid fibril elongation, while high concentration of Aβ40 favors formation of amorphous aggregates. However, there are conflicting reports on this issue. For Aβ42 and Cu(II):Aβ ≤ 1, there is consensus that the lag time is extended upon addition of Cu(II). For Cu(II):Aβ > 1, the lag time is increased upon interaction with Cu(II), and in most cases fibrillation is not observed, presumably because Cu(II) occupies a second more solvent-exposed binding site, which is more prone to form metal ion-bridged species and cause rapid formation of non-fibrillar aggregates. The interesting N-terminally truncated Aβ11-40 with high affinity for Cu(II), exhibits delay of fibrillation upon addition of 0.4 eq. Cu(II). In our view, there are still problems achieving reproducible results in this field, and we provide a shortlist of some of the pitfalls. Finally, we propose a consensus model for the effects of Cu(II) on the aggregation kinetics of Aβ.

In this study, we investigate the conformational characteristics of full-length Aβ 42 peptide monomers in the presence of Na + and Zn 2+ metal ions using atomistic molecular dynamics (MD) simulations with an aim to explore the possible driving forces behind enhanced aggregation rates of the peptides in the presence of salts. The calculations reveal that the presence of metal ions shifts the conformational equilibrium more toward the compact ordered Aβ structures. Such compact ordered structures stabilized by distant nonlocal contacts between two crucial hydrophobic segments, hp1 and hp2, primarily through two important hydrophobic aromatic residues, Phe-19 and Phe-20, are expected to trigger the aggregation process at a faster rate by populating and stabilizing the aggregation prone structures. Formation of a significant number of such distant contacts in the presence of Na + ions has also been found to result in breaking of the N-terminal helix. On the contrary, binding of Zn 2+ ion to Aβ peptide is highly specific, which stabilizes the N-terminal helix instead of breaking it. This explains why the aggregation rate of Aβ peptides is higher in the presence of divalent Zn 2+ ions than monovalent Na + ions. Relatively higher overall stability of the most populated Aβ peptide monomers in the presence of Zn 2+ ions has been found to be associated with specific Zn 2+ -Aβ binding and significant free energy gain.

Alzheimer's disease is one of the most common chronic neurodegenerative disorders. Despite several in vivo and clinical studies, the cause of the disease is poorly understood. Currently, amyloid β (Aβ) peptide and its tendency to assemble into soluble oligomers are known as a main pathogenic event leading to the interruption of synapses and brain degeneration. Targeting neurotoxic Aβ oligomers can help recognize the disease at an early stage or it can be a potential therapeutic approach. Unnatural β-peptidic foldamers are successfully used against many different protein targets due to their favorable structural and pharmacokinetic properties compared to small molecule or protein-like drug candidates. We have previously reported a tetravalent foldamer-dendrimer conjugate which can selectively bind Aβ oligomers. Taking advantage of multivalency and foldamers, we synthesized different multivalent foldamer-based conjugates to optimize the geometry of the ligand. Isothermal titration calorimetry (ITC) was used to measure binding affinity to Aβ, thereafter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based tissue viability assay and impedance-based viability assay on SH-SY5Y cells were applied to monitor Aβ toxicity and protective effects of the compounds. Important factors for high binding affinity were determined and a good correlation was found between influencing the valence and the capability of the conjugates for Aβ binding.

In order to study how acidic pro-peptides inhibit the antimicrobial activity of antimicrobial peptides, we introduce a simple model system, consisting of a 19 amino-acid long antimicrobial peptide, and an N-terminally attached, 10 amino-acid long acidic model pro-peptide. The antimicrobial peptide is a fragment of the crotalicidin peptide, a member of the cathelidin family, from rattlesnake venom. The model pro-peptide is a deca (glutamic acid). Attachment of the model pro-peptide only leads to a moderately large reduction in the binding to- and induced leakage of model liposomes, while the antimicrobial activity of the crotalicidin fragment is completely inhibited by attaching the model pro-peptide. Attaching the pro-peptide induces a conformational change to a more helical conformation, while there are no signs of intra- or intermolecular peptide complexation. We conclude that inhibition of antimicrobial activity by the model pro-peptide might be related to a conformational change induced by the pro-peptide domain, and that additional effects beyond induced changes in membrane activity must also be involved.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, catalyses the conversion of D-glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. While mammalian and yeast GAPDHs are multifunctional proteins that have additional functions beyond those involved in glycolysis, including reactions related to nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis, Escherichia coli GAPDH (ecGAPDH) has only been reported to function in glycolysis. The S-loop of GAPDH is required for interaction with its cofactor and with other proteins. In this study, the three-dimensional crystal structure of GAPDH treated with trehalose is reported at 2.0 Å resolution. Trehalose was used as a cryoprotectant for the GAPDH crystals. The structure of trehalose-bound ecGAPDH was compared with the structures of both NAD + -free and NAD + -bound ecGAPDH. At the S-loop, the bound trehalose in the GAPDH structure induces a 2.4° rotation compared with the NAD + -free ecGAPDH structure and a 3.1° rotation compared with the NAD + -bound ecGAPDH structure., (open access.)

Alzheimer's disease is an irreversible and progressive brain disorder featured by the accumulation of Amyloid-β (Aβ) peptide, which forms insoluble assemblies that builds up into plaques resulting in cognitive decline and memory loss. The formation of fibrillar amyloid deposits is accompanied by conformational changes of the soluble Aβ peptide into β-sheet structures. Strategies to prevent or reduce Aβ aggregation using small molecules such as trehalose have shown beneficial effects under in vitro cell- and in vivo mouse- models. However, the role of trehalose in reducing Aβ peptide aggregation is still not clear. In the present study, using circular dichroism- and fluorescence emission- spectroscopies, we demonstrated that in the presence of trehalose, Aβ peptide adopts more helical content and undergoes a disorder/order conformational transition. Based on our findings, we conclude that trehalose affects the conformation of Aβ peptide to form α-helical structure, which may inhibit the formation of β-sheets and thereby aggregation., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

There are three specific regions in the Amyloid beta (Aβ) peptide sequence where variations cause enhanced toxicity in Alzheimer's disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn 2+ and curcumin on Aβ 40 , and compare these with their previously reported effects on Aβ 42 . Aβ 42 and Aβ 40 differ only near the C-terminus, where curcumin interacts, while Zn 2+ interacts near the N-terminus. Therefore, this comparison should help us differentiate the effect of modulating the C- and the N-termini. We find that curcumin allows fibril-like structures containing the salt bridge to emerge in the mature Aβ 40 aggregates, but not in Aβ 42 . In contrast, we find no difference in the effects of Zn +2 on Aβ 40 and Aβ 42 . In the presence of Zn +2 , both of these fail to form proper fibrils, and the salt bridge remains disrupted. These results indicate that modulations of the Aβ termini can determine the fate of a salt bridge far away in the sequence, and this has significant consequences for Aβ toxicity. We also infer that small molecules can alter oligomer-induced toxicity by modulating the aggregation pathway, without substantially changing the final product of aggregation., (Copyright © 2017. Published by Elsevier Inc.)

The p53 tumor suppressor serves as a major barrier against malignant transformation. Over 50% of tumors inactivate p53 by point mutations in its DNA binding domain. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, misfolded form. The equilibrium between the properly folded and misfolded states of p53 may be affected by molecules that interact with p53, stabilizing its native folding and restoring wild type p53 activity to cancer cells. To select for mutant p53 (mutp53) reactivating peptides, we adopted the phage display technology, allowing interactions between mutp53 and random peptide libraries presented on phages and enriching for phage that favor the correctly folded p53 conformation. We obtained a large database of potential reactivating peptides. Lead peptides were synthesized and analyzed for their ability to restore proper p53 folding and activity. Remarkably, many enriched peptides corresponded to known p53-binding proteins, including RAD9. Importantly, lead peptides elicited dramatic regression of aggressive tumors in mouse xenograft models. Such peptides might serve as novel agents for human cancer therapy.

Aβ(1-40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimer's disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies to control the occurring events. To this purpose the exploitation of small molecules interacting with Aβ aggregation represents one of the possible routes. Moreover, the use specific labeling has represented so far one of the most common and effective methods to investigate such a process. This possibility in turn rests on the reliability of the probe/labels involved. Here we present evidences of the effect of Thioflavin T (ThT), a worldwide used fluorescent dye to monitor amyloid growth, on the Aβ(1-40) conformation, stability and aggregation. By combining experimental information and Molecular Dynamics simulation results, we show that the presence of ThT in solution affects peptide conformation inducing peculiar supramolecular association. In particular ThT interactions with specific Aβ(1-40) residues promote a rigid partially-folded conformation which shifts the balance between different species in solution toward a more aggregation-prone ensemble of peptides, leading to aggregation. Our findings suggest ways for developing strategies to reverse and block aggregation or to stimulate supramolecular assembly and consequently reduce the presence of transient oligomers. This investigation underlines the need of developing label-free techniques for unbiased quantitative studies of Aβ(1-40) aggregation processes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dysfunctional interactions of amyloid-β (Aβ) with Zn and Cu ions are proved to be related to the etiology of Alzheimer's disease (AD). Disruption of these metal-Aβ interactions using metal chelators holds considerable promise as a therapeutic strategy to combat this incurable disease. Herein, we report that two cyclam derivatives (L1 and L2) are capable of modulating Zn(2+)/Cu(2+)-mediated Aβ40 aggregation, reactive oxygen species (ROS) production, and neurotoxicity. These chelators were found to inhibit the metal-induced Aβ40 aggregation, dissociate metal-Aβ40 aggregates and restore the metal-induced β-sheet structure of Aβ40 to its random coil conformation, as observed by BCA protein assay, thioflavin T fluorescence and circular dichroism spectroscopy. Moreover, preliminary investigation of SH-SY5Y cells indicates that L1 and L2 can diminish the neurotoxicity of metal-Aβ40 species, control metal-Aβ40-triggered ROS production and protect cells against apoptosis. These observations warrant the further investigations of L1 and L2 as potential anti-AD agents.

The amyloid-β hypothesis of Alzheimer's Disease (AD) focuses on accumulation of amyloid-β peptide (Aβ) as the main culprit for the myriad physiological changes seen during development and progression of AD including desynchronization of neuronal action potentials, consequent development of aberrant brain rhythms relevant for cognition, and final emergence of cognitive deficits. The aim of this study was to elucidate the cellular and synaptic mechanisms underlying the Aβ-induced degradation of gamma oscillations in AD, to identify aggregation state(s) of Aβ that mediate the peptides neurotoxicity, and to test ways to prevent the neurotoxic Aβ effect. We show that Aβ(1-42) in physiological concentrations acutely degrades mouse hippocampal gamma oscillations in a concentration- and time-dependent manner. The underlying cause is an Aβ-induced desynchronization of action potential generation in pyramidal cells and a shift of the excitatory/inhibitory equilibrium in the hippocampal network. Using purified preparations containing different aggregation states of Aβ, as well as a designed ligand and a BRICHOS chaperone domain, we provide evidence that the severity of Aβ neurotoxicity increases with increasing concentration of fibrillar over monomeric Aβ forms, and that Aβ-induced degradation of gamma oscillations and excitatory/inhibitory equilibrium is prevented by compounds that interfere with Aβ aggregation. Our study provides correlative evidence for a link between Aβ-induced effects on synaptic currents and AD-relevant neuronal network oscillations, identifies the responsible aggregation state of Aβ and proofs that strategies preventing peptide aggregation are able to prevent the deleterious action of Aβ on the excitatory/inhibitory equilibrium and on the gamma rhythm., (Copyright © 2014 the authors 0270-6474/14/3411416-10$15.00/0.)

Mitochondrial dysfunction caused by amyloid β-peptide (Aβ) plays an important role in the pathogenesis of Alzheimer disease (AD). Substantial evidence has indicated that the mitochondrial permeability transition pore (mPTP) opening is involved in Aβ-induced neuronal death and reactive oxygen species (ROS) generation. Astragaloside IV (AS-IV), one of the major active constituents of Astragalus membranaceus, has been reported as an effective anti-oxidant for treating neurodegenerative diseases. However, the molecular mechanisms still need to be clarified. In this study, we investigated whether AS-IV could prevent Aβ1-42-induced neurotoxicity in SK-N-SH cells via inhibiting the mPTP opening. The results showed that pretreatment of AS-IV significantly increased the viability of neuronal cells, reduced apoptosis, decreased the generation of intracellular reactive oxygen species (ROS) and decreased mitochondrial superoxide in the presence of Aβ1-42. In addition, pretreatment of AS-IV inhibited the mPTP opening, rescued mitochondrial membrane potential (ΔΨm), enhanced ATP generation, improved the activity of cytochrome c oxidase and blocked cytochrome c release from mitochondria in Aβ1-42 rich milieu. Moreover, pretreatment of AS-IV reduced the expression of Bax and cleaved caspase-3 and increased the expression of Bcl-2 in an Aβ1-42 rich environment. These data indicate that AS-IV prevents Aβ1-42-induced SK-N-SH cell apoptosis via inhibiting the mPTP opening and ROS generation. These results provide novel insights of AS-IV for the prevention and treatment of neurodegenerative disorders such as AD.

Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. (290)KYCCSRK, which increased IRK V(max) without changing K(m), stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C(292)→A mutant did not stimulate glucose uptake; K(296)→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988-1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C(1056), C(1138), or C(1234), were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia., (© FASEB.)

Abnormal interactions of Zn(2+) and Cu(2+) with the amyloid β-peptide (Aβ) are proposed to play an important role in the neuropathogenesis of Alzheimer's disease (AD). Metal chelators are potential therapeutic agents for AD because they could sequester metals ions from Aβ aggregates and reverse the aggregation. In this study, two nitrogencontaining ligands, TACN and BPA, have been investigated as possible metal chelators in the therapy of Alzheimer's disease. The interactions between the chelators and Aβ40 aggregates are studied by turbidometry, thioflavin T (ThT) fluorescence spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), BCA protein assay, circular dichroism spectroscopy (CD), and atomic force microscopy (AFM). The results demonstrates that TACN and BPA are capable of both disrupting and preventing Zn(2+) or Cu(2+)-induced Aβ40 aggregation. Moreover, they can also suppress the production of H2O2 induced by Cu-Aβ40, associated with toxic oxidative stress in AD.

Diacetyl (DA), an ubiquitous butter-flavoring agent, was found to influence several aspects of amyloid-β (Aβ) aggregation--one of the two primary pathologies associated with Alzheimer's disease. Thioflavin T fluorescence and circular dichroism spectroscopic measurements revealed that DA accelerates Aβ¹⁻⁴² aggregation into soluble and ultimately insoluble β-pleated sheet structures. DA was found to covalently bind to Arg⁵ of Aβ¹⁻⁴² through proteolytic digestion-mass spectrometric experiments. These biophysical and chemical effects translated into the potentiation of Aβ¹⁻⁴² cytotoxicity by DA toward SH-SY5Y cells in culture. DA easily traversed through a MDR1-MDCK cell monolayer, an in vitro model of the blood-brain barrier. Additionally, DA was found not only to be resistant to but also inhibitory toward glyoxalase I, the primary initiator of detoxification of amyloid-promoting reactive dicarbonyl species that are generated naturally in large amounts by neuronal tissue. In light of the chronic exposure of industry workers to DA, this study raises the troubling possibility of long-term neurological toxicity mediated by DA.

The amyloid-β protein (Aβ) oligomers are believed to be the main culprits in the cytoxicity of Alzheimer's disease (AD) and p3 peptides (Aβ17-42 fragments) are present in AD amyloid plaques. Many small-molecule or peptide-based inhibitors are known to slow down Aβ aggregation and reduce the toxicity in vitro, but their exact modes of action remain to be determined since there has been no atomic level of Aβ(p3)-drug oligomers. In this study, we have determined the structure of Aβ17-42 trimers both in aqueous solution and in the presence of five small-molecule inhibitors using a multiscale computational study. These inhibitors include 2002-H20, curcumin, EGCG, Nqtrp, and resveratrol. First, we used replica exchange molecular dynamics simulations coupled to the coarse-grained (CG) OPEP force field. These CG simulations reveal that the conformational ensemble of Aβ17-42 trimer can be described by 14 clusters with each peptide essentially adopting turn/random coil configurations, although the most populated cluster is characterized by one peptide with a β-hairpin at Phe19-Leu31. Second, these 14 dominant clusters and the less-frequent fibril-like state with parallel register of the peptides were subjected to atomistic Autodock simulations. Our analysis reveals that the drugs have multiple binding modes with different binding affinities for trimeric Aβ17-42 although they interact preferentially with the CHC region (residues 17-21). The compounds 2002-H20 and Nqtrp are found to be the worst and best binders, respectively, suggesting that the drugs may interfere at different stages of Aβ oligomerization. Finally, explicit solvent molecular dynamics of two predicted Nqtrp-Aβ17-42 conformations describe at atomic level some possible modes of action for Nqtrp.

The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we studied the effect of two of these factors--the magnesium ion cofactor and the protein substrate--on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of adenosine triphosphate (ATP) competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also showed that the peptidic substrate affects the potency of these inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during drug discovery.

To identify a lead skeleton structure for optimization of scyllo-inositol-based inhibitors of amyloid-beta peptide (Aβ) aggregation, we have synthesized aldoxime, hydroxamate, carbamate, and amide linked scyllo-inositol derivatives. These structures represent backbones that can be readily expanded into a wide array of derivatives. They also provide conservative modifications of the scyllo-inositol backbone, as they maintain the display of the equatorial polar atoms, preserving the stereochemical requirement necessary for maximum inhibition of Aβ(1-42) fiber formation. In addition, a reliable work plan for screening derivatives was developed in order to preferentially identify a backbone(s) structure that prevents fibrillogenesis and stabilizes nontoxic small molecular weight oligomers, as we have previously reported for scyllo-inositol. In the present studies, we have adapted a high throughput ELISA-based oligomerization assay followed by atomic force microscopy to validate the results screen compounds. The lead compounds were then tested for toxicity and ability to rescue Aβ(1-42) induced toxicity in vitro and the affinity of the compounds for Aβ(1-42) compared by mass spectrometry. The data to suggest that compounds must maintain a planar conformation to exhibit activity similar to scyllo-inositol and that the oxime derivative represents the lead backbone for future development.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of β-sheet-rich amyloid oligomers or fibrils which are associated with cellular toxicity in the brain. Inhibition of Aβ aggregation could be a viable therapeutic strategy for slowing and/or preventing the progress of AD. Here we reported that α-mangostin (α-M), a polyphenolic xanthone derivative from mangosteen, concentration-dependently attenuated the neurotoxicity induced by Aβ-(1-40) or Aβ-(1-42) oligomers (EC(50) = 3.89 nM, 4.14 nM respectively) as observed by decreased cell viability and impaired neurite outgrowth in primary rat cerebral cortical neurons. Molecular docking and dynamics simulations demonstrated that α-M could potentially bind to Aβ and stabilize α-helical conformation. α-M was found to directly dissociate Aβ-(1-40) and Aβ-(1-42) oligomers by blotting with oligomer-specific antibodies. ThioflavinT fluorescence assay and electron microscopy imaging further demonstrated that α-M blocked the fibril formation as well as disturbed the pre-formed fibrils. Taken together, our results indicate that α-M is capable to inhibit and dissociate the Aβ aggregation, which could contribute to its effect of attenuating Aβ oligomers-induced neurotoxicity. Thus, α-M could be a great potential candidate for AD treatment. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.

The loss of metal homeostasis and the toxic effect of metal ion are important events in neurodegenerative and age-related diseases, such as Alzheimer's disease (AD). For the first time, we investigated the impacts of mercury(II) ions on the folding and aggregation of Alzheimer's tau fragment R2 (residues 275-305: VQIIN KKLDL SNVQS KCGSK DNIKH VPGGGS), corresponding to the second repeat unit of the microtubule-binding domain, which was believed to be pivotal to the biochemical properties of full tau protein. By ThS fluorescence assay and electron microscopy, we found that mercury(II) dramatically promoted heparin-induced aggregation of R2 at an optimum molar ratio of 1: 2 (metal: protein), and the resulting R2 filaments became smaller. Isothermal titration calorimetry (ITC) experiment revealed that the strong coordination of mercury(II) with R2 was an enthalpy-controlled, entropy-decreased thermodynamic process. The exceptionally large magnitude of heat release (ΔH₁ = -34.8 Kcal mol⁻¹) suggested that the most possible coordinating site on the R2 peptide chain was the thiol group of cysteine residue (Cys291), and this was further confirmed by a control experiment using Cys291 mutated R2. Circular dichroism spectrum demonstrated that this peptide underwent a significant conformational change from random coil to β-turn structure upon its binding to mercury(II) ion. This study was undertaken to better understand the mechanism of tau aggregation, and evaluate the possible role of mercury(II) in the pathogenesis of AD., (© 2010 Wiley Periodicals, Inc.)

In this work, we studied how an amphipathic peptide of the surface of the globular protein thioredoxin, TRX94-108, acquires a native-like structure when it becomes involved in an apolar interaction network. We designed peptide variants where the tendency to form alpha-helical conformation is modulated by replacing each of the leucine amino acid residues by an alanine. The induction of structure caused by sodium dodecyl sulfate (SDS) binding was studied by capillary zone electrophoresis, circular dichroism, DOSY-NMR, and molecular dynamics simulations (MDS). In addition, we analyzed the strength of the interaction between a C18 RP-HPLC matrix and the peptides. The results presented here reveal that (a) critical elements in the sequence of the wild-type peptide stabilize a SDS/peptide supramolecular cluster; (b) the hydrophobic nature of the interaction between SDS molecules and the peptide constrains the ensemble of conformations; (c) nonspecific apolar surfaces are sufficient to stabilize peptide secondary structure. Remarkably, MDS shed light on a contact network formed by a limited number of SDS molecules that serves as a structural scaffold preserving the helical conformation of this module. This mechanism might prevail when a peptide with low helical propensity is involved in structure consolidation. We suggest that folding of peptides sharing this feature does not require a preformed tightly-packed protein core. Thus, the formation of specific tertiary interactions would be the consequence of peptide folding and not its cause. In this scenario, folding might be thought of as a process that includes unspecific rounds of structure stabilization guiding the protein to the native state., (2010 Wiley-Liss, Inc.)

The tetrameric M2 proton channel from influenza A virus conducts protons at low pH and is inhibited by aminoadamantyl drugs such as amantadine and rimantadine (Rmt). We report magic angle spinning NMR spectra of POPC and DPhPC membrane-embedded M2(18-60), both apo and in the presence of Rmt. Similar line widths in the spectra of apo and bound M2 indicate that Rmt does not have a significant impact on the dynamics or conformational heterogeneity of this construct. Substantial chemical shift changes for many residues in the transmembrane region support an allosteric mechanism of inhibition. An Rmt titration supports a binding stoichiometry of >1 Rmt molecule per channel and shows that nonspecific binding or changes in membrane composition are unlikely sources of the chemical shift changes. In addition, doubling of spectral lines in all of the observed samples provides evidence that the channel assembles with twofold symmetry.

A key event in Alzheimer's disease (AD) is age-dependent, brain accumulation of amyloid beta-protein (Abeta) leading to Abeta self-association into neurotoxic oligomers. Previously, we showed that Abeta oligomerization and neurotoxicity could be inhibited by C-terminal fragments (CTFs) of Abeta42. Because these CTFs are highly hydrophobic, we asked if they themselves aggregated and, if so, what parameters regulated their aggregation. To answer these questions, we investigated the dependence of CTF aqueous solubility, aggregation kinetics, and morphology on peptide length and sequence and the correlation between these characteristics and inhibition of Abeta42-induced toxicity. We found that CTFs up to 8 residues long were soluble at concentrations >100 microM and had a low propensity to aggregate. Longer CTFs were soluble at approximately 1-80 microM, and most, but not all, readily formed beta-sheet-rich fibrils. Comparison to Abeta40-derived CTFs showed that the C-terminal dipeptide I41-A42 strongly promoted aggregation. Aggregation propensity correlated with the previously reported tendency to form beta-hairpin conformation but not with inhibition of Abeta42-induced neurotoxicity. The data enhance our understanding of the physical characteristics that affect CTF activity and advance our ability to design, synthesize, and test future generations of inhibitors.

Accumulating evidence suggests that Abeta(1-42)-membrane interactions may play an important role in the pathogenesis of Alzheimer's disease. However, the mechanism of this structural transition remains unknown. In this work, we have shown that submicellar concentrations of sodium dodecyl sulfate (SDS) can provide a minimal platform for Abeta(1-42) self-assembly. To further investigate the relation between Abeta(1-42) structure and function, we analyzed peptide conformation and aggregation at various SDS concentrations using circular dichroism (CD), Fourier transform infrared spectroscopy, and gel electrophoresis. These aggregates, as observed via atomic force microscopy, appeared as globular particles in submicellar SDS with diameters of 35-60 nm. Upon sonication, these particles increased in disc diameter to 100 nm. Pyrene I (3)/I (1) ratios and 1-anilinonaphthalene-8-sulfonic acid binding studies indicated that the peptide interior is more hydrophobic than the SDS micelle interior. We have also used Forster resonance energy transfer between N-terminal labeled pyrene and tyrosine (10) of Abeta(1-42) in various SDS concentrations for conformational analysis. The results demonstrate that SDS at submicellar concentrations accelerates the formation of spherical aggregates, which act as niduses to form large spherical aggregates upon sonication.

Septic or endotoxic shock is a common cause of death in hospital intensive care units. In the last decade numerous antimicrobial peptides and proteins have been tested in the search for an efficient drug to treat this lethal disease. Now in phase III clinical trials, rBPI(21), a recombinant N-terminal fragment of the bactericidal/permeability-increasing protein (BPI), is a promising drug to reduce lesions caused by meningococcal sepsis. We correlated structural and stability data with functional information of rBPI(21) bound to both model systems of eukaryotic and bacterial membranes. On interaction with membranes, rBPI(21) loses its conformational stability, as studied by circular dichroism. This interaction of rBPI(21) at membrane level was higher in the presence of negatively charged phospholipid relatively to neutral ones, with higher partition coefficients (K(p)), suggesting a preference for bacterial membranes over mammalian membranes. rBPI(21) binding to membranes is reinforced when its disulfide bond is broken due to conformational changes of the protein. This interaction is followed by liposome aggregation due to unfolding, which ensures protein aggregation, and interfacial localization of rBPI(21) in membranes, as studied by extensive quenching by acrylamide and 5-deoxylstearic acid and not by 16-deoxylstearic acid. An uncommon model of the selectivity and mechanism of action is proposed, where membrane induces unfolding of the antimicrobial protein, rBPI(21). The unfolding ensures protein aggregation, established by protein-protein interaction at membrane surface or between adjacent membranes covered by the unfolded protein. This protein aggregation step may lead to membrane perturbation.

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of a misfolded form (PrP(Sc)) of the cellular prion protein (PrP(C)) in the brains of affected individuals. The conversion of PrP(C) to PrP(Sc) is thought to involve a change in protein conformation from a normal, primarily alpha-helical structure into a beta-sheet conformer. Few proteins have been identified that differentially interact with the two forms of PrP. It has been reported that plasminogen binds to PrP(Sc) from a variety of prion phenotypes. We have examined potential motifs within the kringle region that may be responsible for binding to PrP. We synthesized 12-15-mer peptides that contain small, repetitive stretches of amino acid residues found within the kringle domains of plasminogen. These synthetic peptides were found to capture PrP(Sc) from the brain homogenates of bovine spongiform encephalopathy affected cattle, chronic wasting disease affected elk, experimental scrapie of hamsters and that of subjects affected by Creutzfeldt-Jakob disease, without binding to PrP(C) in unaffected controls. Therefore, we have identified critical peptide motifs that may be important for protein-protein interactions in prion disease pathogenesis. The ability of these synthetic peptides to bind preferentially to PrP(Sc) suggests a potential application in the diagnosis of prion diseases.

Mastoparan-X, a 14-residue peptide found in wasp venom, does not adopt a well-defined structure in water, but it folds into an alpha-helix upon addition of trifluoroethanol (TFE). At low levels of TFE, the peptide is partially folded, passing through intermediate stages of folding as the amount of TFE is increased. These partially folded states have been characterized by CD and NMR spectroscopy, and methods to estimate the helical content from CD, chemical shift, and nuclear overhauser effect (NOE) data are compared. Variation in the sign and intensity of NOE cross-peaks is observed in different regions of the peptide, indicative of greater mobility of the sidechains compared to the backbone of the peptide. Furthermore, variation in the sidechain mobility is observed, both between sidechains of different amino acids and within the sidechain of a given amino acid. By monitoring chemical shifts and NOE intensities as the TFE concentration is increased, the initiation site for helix formation could be identified. Furthermore, details of the peptide structure and dynamics during the folding process were elucidated., (2007 Wiley Periodicals, Inc)

Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-beta-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions--perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including alpha B-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.

Alzheimer's disease (AD) is characterized by the aggregation of misfolded proteins. Previously we reported activation of the unfolded protein response (UPR) in AD neurons. A potential source for UPR activation in AD neurons may be the increased levels of beta-amyloid (Abeta). In this study, we used preparations enriched in oligomeric or fibrillar Abeta (1-42) to investigate the role of the conformational state of Abeta in UPR activation in differentiated neuroblastoma cells. Both oligomeric and fibrillar Abeta (1-42) do not induce BiP expression to the extent that it can be detected in a pool of cells. However, using a fluorescent UPR reporter cell line that allows analysis of individual cells, we demonstrated mild activation of the UPR by oligomeric but not fibrillar Abeta (1-42). We showed that oligomeric Abeta (1-42) is significantly more toxic to cells primed for UPR than is fibrillar Abeta (1-42), indicating that activation of the UPR contributes to oligomer-specific Abeta (1-42) toxicity. Because UPR activation is observed in AD brain at a stage that precedes the massive fibrillar Abeta deposition and tangle formation, this may indicate a role for nonfibrillar Abeta in the induction of the UPR in AD neurons.

Copper (II) has been implicated in the pathology of Alzheimer's disease (AD) for the impaired homeostatic mechanism found in the brains of AD patients. Here we studied the binding properties of Cu(II) with the first microtubule-binding repeat, encompassing residues 256-273 of the human tau441 sequence. Additionally, the effect of Cu(II) on the assembly of this repeat was also investigated. Our results indicate that Cu(II) can bind to this repeat with His(268) involved and has an inhibiting effect on the in vitro aggregation of this repeat. This work provides new insight into the role of Cu(II) in Alzheimer's disease.

P-Glycoprotein (P-gp, ABCB1) transports a variety of structurally unrelated cytotoxic compounds out of the cell. Each homologous half of P-gp has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD) and is joined by a linker region. It has been postulated that binding of two ATP molecules at the NBD interface to form a "nucleotide sandwich" induces drug efflux by altering packing of the TM segments that make up the drug-binding pocket. To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. It was found that ATP binding alone could alter disulfide cross-linking between the TM segments. For example, ATP inhibited cross-linking of mutant L339C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) but promoted cross-linking of mutant F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Cross-linking of some mutants, however, appeared to require ATP hydrolysis as introduction of the catalytic carboxylate mutations into mutant L332C(TM6)/L975C(TM12) inhibited ATP-dependent cross-linking. Cross-linking between cysteines in the TM segments also could be altered via introduction of a single catalytic carboxylate mutation into mutant L332C(TM6)/L975C(TM12) or by using the nonhydrolyzable ATP analogue, AMP.PNP. The results show that the TM segments are quite sensitive to changes within the ATP-binding sites because different conformations could be detected in the presence of ATP, AMP.PNP, during ATP hydrolysis or through mutation of the catalytic carboxylates.

Conformational changes in the prion protein cause transmissible spongiform encephalopathies, also referred to as prion diseases. In its native state, the prion protein is innocuous (PrPC), but it can misfold into a neurotoxic and infectious isoform (PrPSc). The full-length cellular form of the prion protein consists of residues 23-230, with over half of the sequence belonging to the unstructured N-terminal domain and the remaining residues forming a small globular domain. During misfolding and aggregation, portions of both the structured and unstructured domains are incorporated into the aggregates. After limited proteolysis by proteinase K, the most abundant fragment from brain-derived prion fibrils is a 141-residue fragment composed of residues 90-230. Here we describe simulations of this fragment of the human prion protein at low pH, which triggers misfolding, and at neutral pH as a control. The simulations, in agreement with experiment, show that this biologically and pathologically relevant prion construct is stable and native-like at neutral pH. In contrast, at low pH the prion protein is destabilized via disruption of critical long-range salt bridges. In one of the low pH simulations this destabilization resulted in a conformational transition to a PrPSc-like isoform consistent with our previous simulations of a smaller construct.

The aggregation (fibril formation) of amyloid beta-protein (Abeta) is considered to be a crucial step in the etiology of Alzheimer's disease (AD). The inhibition of Abeta aggregation and/or decomposition of fibrils formed in aqueous solution by small compounds have been studied extensively for the prevention and treatment of AD. However, recent studies suggest that Abeta aggregation also occurs in lipid rafts mediated by a cluster of monosialoganglioside GM1. This study examined the effects of representative compounds on Abeta aggregation and fibril destabilization in the presence of GM1-containing raft-like liposomes. Among the compounds tested, nordihydroguaiaretic acid (NDGA), rifampicin (RIF), tannic acid (TA), and quercetin (QUE) showed strong fibrillization inhibitory activity. NDGA and RIF inhibited the binding of Abeta to GM1 liposomes by competitively binding to the membranes and/or direct interaction with Abeta in solution, thus at least partly preventing fibrils from forming. Coincubation of Abeta with NDGA, RIF, and QUE in the presence of GM1 liposomes resulted in elongate particles, whereas the presence of TA yielded protofibrillar structures. TA and RIF also destabilized fibrils. The most potent NDGA prevented Abeta-induced toxicity in PC12 cells by inhibiting Abeta accumulation. Furthermore, a comparison of the inhibitory effects of various compounds between aqueous-phase and GM1-mediated aggregation of Abeta suggested that the two aggregation processes are not identical.

Trifluoroethanol (TFE)-induced conformational changes in dynorphin A (1-13) were investigated using charge-state distribution (CSD) and hydrogen-deuterium exchange (HDX), combined with electrospray ionization (ESI) mass spectrometry (MS). Individual amino acids involved in secondary structural elements were identified by collision-induced dissociation-tandem mass spectrometry (MS/MS). It was observed that dynorphin A (1-13) largely exists in an unfolded conformation and a folded structure in increasing concentrations of TFE. In 50% TFE, it forms an alpha-helix that encompasses residues 1-9 and remains flexible from residues 10 to 13.

Employing high-resolution (13)C solution NMR and circular dichroism (CD) spectroscopic techniques, the distinctive influence of two intimately related hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and hexafluoroacetone trihydrate (HFA), on the structural characteristics of Bombyx mori (B. mori) silk fibroin, the chymotrypsin precipitate (C(p)) fraction, and two synthetic peptides, (AGSGAG)(5) and (AG)(15), is described. The observed (13)C solution NMR and CD spectra of these polypeptides in HFIP and HFA revealed a distinctive influence on their conformational characteristics. The (13)C NMR spectra, as analyzed from the unique chemical shifts of C(alpha) and C(beta) resonances of constituent residues revealed that fibroin largely assumes helical conformation(s) in both solvents. However, the peak shifts were greater for the samples in HFIP, indicating that the types of helical structure(s) may be different from the one populated in HFA. Similar structural tendencies of these polypeptides were reflected in CD spectra. The observed CD patterns, i.e., a strong positive band at approximately 190 nm and negative bands at approximately 206 and 222 nm, have been attributed to the preponderance of helical structures. Of the two prevalent helical structures, alpha-helix and 3(10)-helix, the evidence emerged for the fibroin protein in favor of 3(10)-helical structure stabilization in HFIP and its significant disruption in HFA, as deduced from the characteristic R1 (=[theta](190)/[theta](202)) and R2 (=[theta](222)/[theta](206)) ratios, determined from the CD data. Conversely, the native polypeptides and synthetic peptide fragments derived from highly crystalline regions of the silk fibroin protein sustained predominantly an unordered structure in HFA solvent.

Four sites of the non-homologous region (coding amino acid residues of 347, 421, 466 and 533) of a gene were randomly selected for splitting to investigate the function of beta-glucosidase from Agrobacterium tumefaciens in the co-refolding of peptides into the catalytically active enzyme. As a result of gene splitting, four N- and C-terminal domain peptides were obtained as insoluble inclusion bodies. No catalytic activity was observed when these fragments refolded individually. However, a considerable amount of activity was restored when the two fragments derived from N- and C- terminal peptides were co-refolded together. The deletion of amino acid residues in the non-homologous region resulted in a complete loss of enzyme activity, which suggests that truncation of amino acids in this region strongly affects the co-refolding ability of the enzyme to maintain activity.

Recent reports suggest that some commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) unexpectedly shift the cleavage products of amyloid precursor protein (APP) to shorter, less fibrillogenic forms, although the underlying mechanism remains unknown. We now demonstrate, using a fluorescence resonance energy transfer method, that Abeta(42)-lowering NSAIDs specifically affect the proximity between APP and presenilin 1 and alter presenilin 1 conformation both in vitro and in vivo, suggesting a novel allosteric mechanism of action.

The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status. Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with NifL under conditions unfavorable for nitrogen fixation. We have recently shown that the NifL-NifA system responds directly to physiological concentrations of 2-oxoglutarate, resulting in relief of NifA activity from inhibition by NifL under conditions when fixed nitrogen is limiting. The inhibitory activity of NifL is restored under conditions of excess combined nitrogen through the binding of the signal transduction protein Av GlnK to the carboxyl-terminal domain of NifL. The amino-terminal domain of NifA comprises a GAF domain implicated in the regulatory response to NifL. A truncated form of NifA lacking this domain is not responsive to 2-oxoglutarate in the presence of NifL, suggesting that the GAF domain is required for the response to this ligand. Using isothermal titration calorimetry, we demonstrate stoichiometric binding of 2-oxoglutarate to both the isolated GAF domain and the full-length A. vinelandii NifA protein with a dissociation constant of approximately 60 microm. Limited proteolysis experiments indicate that the binding of 2-oxoglutarate increases the susceptibility of the GAF domain to trypsin digestion and also prevents NifL from protecting these cleavage sites. However, protection by NifL is restored when the non-modified (non-uridylylated) form of Av GlnK is also present. Our results suggest that the binding of 2-oxoglutarate to the GAF domain of NifA may induce a conformational change that prevents inhibition by NifL under conditions when fixed nitrogen is limiting.

Glutamate 681 is thought to be located within the transport channel of band 3 (AE1, the chloride/bicarbonate exchanger), where it acts as a proton donor for the anion/proton cotransport function. Here we show that neutralization of the negative charge on glutamate 681 by chemically modifying band 3 with Woodward's reagent K plus sodium borohydride (i.e., the modification process) exposes a cryptic, conformationally active chloride-binding site which functions to modulate allosterically the conformational state of the band 3 dimer. Chloride binding was determined by measuring the effect of increasing chloride concentration on the rate of DBDS (4,4'-dibenzamido-2,2'-stilbenedisulfonate) release from band 3 using a stopped-flow fluorescence kinetic inhibitor replacement assay with DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) as the replacing inhibitor. The time course for DBDS release from unmodified, control band 3 was monophasic and exponential. Chloride binding to the transport site accelerated the rate of DBDS release, with the observed rate constant showing a hyperbolic dependence on chloride concentration, while the total change in reaction fluorescence remained constant. After modification of glutamate 681, DBDS release was monophasic in the absence of chloride, but the rapid addition of chloride at constant ionic strength induced a doubling in the fluorescence quantum yield for the bound DBDS molecules. This was associated with the development of 50:50 biphasic kinetics for DBDS release. Such changes were independent of the degree of modification of the band 3 subunit population between the 66% and 91% levels. Titration of the increase in total reaction fluorescence gave an apparent chloride binding K(d) of between 7 and 10 mM, which is 25-40-fold higher in affinity than chloride binding to the transport site. The dependence of the kinetic constants for both phases of the DBDS release reaction on chloride concentration was nonhyperbolic, which contrasts with unmodified band 3, and is indicative of the presence of two classes of chloride-binding sites on the modified transporter. We have also found that the fraction of subunits capable of binding DBDS reversibly, or DIDS covalently, decreased nonlinearly in the absence of chloride as the level of modification of the band 3 subunit population increased. In contrast, the same DBDS binding correlation plot showed a maximum in the presence of saturating chloride. The observation of such nonlinear correlation plots is consistent with a noncooperative dimer model for the modification process, where each dimeric species must possess different properties with respect to stilbenedisulfonate binding capacity and with respect to the spectral-kinetic response of bound stilbenedisulfonate molecules to the addition of chloride. Within the context of this model, the fractions of the three molecular dimeric species (i.e., the unmodified dimer, the dimer with one subunit modified, and the fully modified band 3 dimer) are calculated as a function of the level of modification of the band 3 subunit population. Nonlinear correlation plots are generated by then assigning the following specific properties to each dimeric species. The unmodified dimer binds DBDS but does not change its fluorescence quantum yield upon addition of chloride. The half-modified dimer binds DBDS on both modified and unmodified subunits, and both of those DBDS molecules increase their fluorescence quantum yield by 2-fold when chloride is added, and the system develops 50:50 biphasic DBDS release kinetics. Finally, the model requires that the fully modified dimer does not bind DBDS or DIDS. This model generates theoretical correlation plots that can represent the data presented in this study. We propose that neutralization of glutamate 681 on the half-modified band 3 dimer exposes an allosteric, chloride-binding modifier site which functions to facilitate the anion/proton cotransport process (a) by blocking the "redocking" of the carboxyl side chain of glutamate (thus raising its pK) and (b) by inducing amate (thus raising its pK) and (b) by inducing a conformational change in the band 3 dimer from a symmetrical to an asymmetrical state.

The interaction of A beta peptides with the lipid matrix of neuronal cell membranes plays an important role in the pathogenesis of Alzheimer's disease. By using EPR and CD spectroscopy, we found that in the presence of Cu(2+) or Zn(2+), pH, cholesterol, and the length of the peptide chain influenced the interaction of these peptides with lipid bilayers. In the presence of Zn(2+), A beta 40 and A beta 42 both inserted into the bilayer over the pH range 5.5-7.5, as did A beta 42 in the presence of Cu(2+). However, A beta 40 only penetrated the lipid bilayer in the presence of Cu(2+) at pH 5.5-6.5; at higher pH there was a change in the Cu(2+) coordination sphere that inhibited membrane insertion. In the absence of the metals, insertion of both peptides only occurred at pH < 5.5. Raising cholesterol to 0.2 mol fraction of the total lipid inhibited insertion of both peptides under all conditions investigated. Membrane insertion was accompanied by the formation of alpha-helical structures. The nature of these structures was the same irrespective of the conditions used, indicating a single low energy structure for A beta in membranes. Peptides that did not insert into the membrane formed beta-sheet structures on the surface of the lipid.

Alzheimer's disease is the commonest form of senile dementia, affecting almost 20 million people worldwide. This neurodegenerative disorder is characterized by amyloid deposition in senile plaques, composed primarily of fibrils of an aggregated peptide, beta-amyloid. Fibrillation of beta-amyloid is a nucleation-dependent polymerization process, which is controlled by two kinetics parameters: the nucleation rate and the elongation or growth rate. As the kinetics of fibrillation is strongly dependent on the presence of trace amounts of fibrils, we suggest that the aggregation of beta-amyloid is a model of autocatalytic reaction. A mathematical analysis, permitting quantitative monitoring of the kinetics of fibrillogenesis of beta-amyloid, nucleation, and elongation constants, is presented. The model was checked by applying it to the aggregation of the fragment 1-40 of the beta-amyloid. Understanding of these rate constants may facilitate the study of the effect of substances used for controlling fibril creation and growth. The disaggregating effect of dodecyl trimethylammonium bromide, a cationic surfactant, was easily quantified by means of the model., (Copyright 2003 Wiley Periodicals, Inc.)

Nuclear magnetic resonance (NMR) spectroscopy reveals that higher-order aggregates of glucagon-like peptide-1-(7-36)-amide (GLP-1) in pure water at pH 2.5 are disrupted by 35% 2,2,2-trifluoroethanol (TFE), and form a stable and highly symmetric helical self-aggregate. NMR spectra show that the helical structure is identical to that formed by monomeric GLP-1 under the same experimental conditions [Chang et al., Magn. Reson. Chem. 37 (2001) 477-483; Protein Data Bank at RCSB code: 1D0R], while amide proton exchange rates reveal a dramatic increase of the stability of the helices of the self-aggregate. Pulsed-field gradient NMR diffusion experiments show that the TFE-induced helical self-aggregate is a dimer. The experimental data and model calculations indicate that the dimer is a parallel coiled coil, with a few hydrophobic residues on the surface that may cause aggregation in pure water. The results suggest that the coiled coil dimer is an intermediate state towards the formation of higher aggregates, e.g. fibrils.

Integration of HIV-1 genome into host cell chromosome is mediated by viral integrase (IN). The IN catalytic core (CC, IN(50-212)) dimerizes through mutual interactions of its alpha1 and alpha5 helices. Peptides INH1 and INH5 reproducing these helix sequences strongly inhibited IN. For instance, an IC(50) of 80 nM was determined for INH5 in integration assays using wild-type IN (wtIN). In size exclusion chromatography, INH1 and INH5 perturbed the association-dissociation equilibrium of both dmIN (IN(1-288)/F185K/C280S) and CC, leading to monomers as surviving species, while in circular dichroism, binding of peptides to dmIN altered the protein conformation. Thus, enzyme deactivation, subunit dissociation, and protein unfolding are events which parallel one another. The target of INH5 in the enzyme was then identified. In fluorescence spectroscopy, C(0.5) values of 168 and 44 nM were determined for the binding affinity of INH5 to IN and CC, respectively, at 115 nM subunit concentration, while interaction of INH5 with INH1 was found stronger than interaction of INH5 with itself (23 times larger in term of dissociation constants). These results strongly suggested that the alpha1 helix is the privileged target of INH5. The latter could serve as a lead for the development of new chemotherapeutic agents against HIV-1.

Human high molecular mass kininogen (HK) participates as nonenzymatic cofactor in the contact system. Here, we show that recombinant domain D5 of HK (rD5) prolongs the clotting time of the intrinsic pathway of coagulation and attenuates the generation of bradykinin. Further studies indicate that a correct fold of domain D5 within HK is required for the activation of the contact system. The folding of rD5 seems to be modulated by the metal ions Zn2+, Ni2+, and Cu2+ as a specific antibody directed against the zinc-binding site in HK binds to HK and rD5 in a metal ion concentration dependent manner. The finding that these three metal ions specifically affect contact activation suggests that they regulate the accessibility of rD5 for negatively charged surfaces. Support for the assumption that the observed phenomena are due to conformational changes was obtained by fluorescence spectroscopy of rD5, demonstrating that its fluorescence spectrum was changed in the presence of ZnCl2. Moreover, negative staining electron microscopy experiments suggest that the zinc-induced changes in D5 also affect the conformation of the entire HK protein. The present data emphasize the role of zinc and other metal ions in the regulation of contact activation.