11 research outputs found

    Determination of Secondary Structure of Proteins by Nanoinfrared Spectroscopy

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    Nanoscale infrared spectroscopy (AFMIR) is becoming an important tool for the analysis of biological sample, in particular protein assemblies, at the nanoscale level. While the amide I band is usually used to determine the secondary structure of proteins in Fourier transform infrared spectroscopy, no tool has been developed so far for AFMIR. The paper introduces a method for the study of secondary structure of protein based on a protein library of 38 well-characterized proteins. Ascending stepwise linear regression (ASLR) and partial least square (PLS) regression were used to correlate spectrum characteristic bands with the major secondary structures (α-helixes and β-sheets). ASLR appears to provide better results than PLS. The secondary structure predictions are characterized by a root mean square standard error in a cross validation of 6.39% for α-helixes and 6.23% for β-sheets

    ATR-FTIR spectra of Aβ(1–40) and Aβ(1–40)E22G.

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    <p>FTIR spectra of Aβ(1–40) and Aβ(1–40)E22G were taken in the presence and in the absence of added Ca<sup>2+</sup>, showing the amide I region of the spectra (1600–1700 cm<sup>−1</sup>). Aliquots of 2 µl were taken from each sample at <i>t</i> = 0, 2, 6, 24, 48, 72, and 96 h (shown in blue, green, red, cyan, purple, mustard, and dark blue, respectively). The data shown here were collected in one continuous experiment and are representative of three independent trials.</p

    Oligomers and fibrils formation differentiated by ThT fluorescence.

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    <p>ThT fluorescence intensity was monitored to follow fibrillogenesis of Aβ(1–40) and Aβ(1–40)E22G in the presence and in the absence of 2 mM Ca<sup>2+</sup>. Black bars, Aβ(1–40) in phosphate buffer (“–Ca<sup>2+</sup> condition”); light grey bars, Aβ(1–40) in 2 mM CaCl<sub>2</sub>; dark grey bars, Aβ(1–40)E22G in phosphate buffer; light blue bars, Aβ(1–40)E22G in CaCl<sub>2</sub>. Shown are averages of values obtained in four independent experiments; error bars indicating the standard error of the average.</p

    Morphological comparison of Aβ(1–40) and Aβ(1–40)E22G.

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    <p>Contact mode AFM images (5 µm × 5 µm, Z scale 15 nm) of Aβ(1–40) and Aβ(1–40)E22G peptides on mica, recorded either in phosphate buffer or in MOPS buffer with Ca<sup>2+</sup>. Samples of Aβ(1–40) and Aβ(1–40)E22G in the presence and absence of added Ca<sup>2+</sup> (marked as “+Ca<sup>2+</sup>” or “−Ca<sup>2+</sup>”, respectively) at <i>t</i> = 0, 6, or 72 h. Closer views (1 µm × 1 µm, Z scale 15 nm) of oligomers, protofibrils and fibrils are shown as insets in the panel of <i>t</i> = 72 h (C, F, I, L). Images A, D, G, J were taken at <i>t</i> = 0; images B, E, H, K were taken at <i>t</i> = 6 h. Peptide concentration was the same in all samples.</p

    Chemical Cross-Linking/Mass Spectrometry Maps the Amyloid β Peptide Binding Region on Both Apolipoprotein E Domains

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    Apolipoprotein E (apoE) binds the amyloid β peptide (Aβ), one of the major culprits in Alzheimer’s disease development. The formation of apoE:Aβ complexes is implicated in both Aβ clearance and fibrillization. However, the binding interface between apoE and Aβ is poorly defined despite substantial previous research efforts, and the exact role of apoE in the pathology of Alzheimer’s disease remains largely elusive. Here, we compared the three main isoforms of apoE (E2, E3, and E4) for their interaction with Aβ<sub>1–42</sub> in an early stage of aggregation and at near physiological conditions. Using electron microscopy and Western blots, we showed that all three isoforms are able to prevent Aβ fibrillization and form a noncovalent complex, with one molecule of Aβ bound per apoE. Using chemical cross-linking coupled to mass spectrometry, we further examined the interface of interaction between apoE2/3/4 and Aβ. Multiple high-confidence intermolecular apoE2/3/4:Aβ cross-links confirmed that Lys16 is located in the region of Aβ binding to apoE2/3/4. Further, we demonstrated that both N- and C-terminal domains of apoE2/3/4 are interacting with Aβ. The cross-linked sites were mapped onto and evaluated in light of a recent structure of apoE. Our results support binding of the hydrophobic Aβ at the apoE domain–domain interaction interface, which would explain how apoE is able to stabilize Aβ and thereby prevent its subsequent aggregation

    Mechanism of regulation and activation of lipidated apoE4.

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    <p>After encounter of lipids, two free apoE4 molecules will adopt either a compact hairpin (A) or opened hairpin (B) configuration around the nanodisc which is followed/concomitant with an elongation of NT helix 4 (C and D). Conversions of the two configurations are possible. On the opened hairpin configuration, the elongated NT helix 4 is accessible and recognized by the LDL receptors (D). The helices are colored as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006165#pcbi.1006165.g003" target="_blank">Fig 3</a>. The elongated NT helix 4 is highlighted in dashed green.</p

    Lys-Lys residues cross-linked in apoE4 nanodiscs and validation of the opened hairpin and compact hairpin models by XL distance compatibility<sup>a</sup>.

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    <p>Lys-Lys residues cross-linked in apoE4 nanodiscs and validation of the opened hairpin and compact hairpin models by XL distance compatibility<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006165#t001fn001" target="_blank"><sup>a</sup></a>.</p

    Structural models of apoE4 at the surface of a nanodisc.

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    <p>The structural modeling produces two different models of monomeric lipidated apoE4: the opened hairpin (A) and the compact hairpin (B) models that fulfill 12 and 19 out of 22 experimental XL restraints, respectively. For each model two views are shown, from the top of the lipid disc (upper) and rotated by 90° reflecting a view from the inside of the lipid bilayer of the nanodisc (lower). Cross-links with satisfied distance restraints within their respective models are shown as black lines. The secondary structure elements are colored as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006165#pcbi.1006165.g003" target="_blank">Fig 3</a>.</p

    Lipidated apolipoprotein E4 structure and its receptor binding mechanism determined by a combined cross-linking coupled to mass spectrometry and molecular dynamics approach

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    <div><p>Apolipoprotein E (apoE) is a forefront actor in the transport of lipids and the maintenance of cholesterol homeostasis, and is also strongly implicated in Alzheimer’s disease. Upon lipid-binding apoE adopts a conformational state that mediates the receptor-induced internalization of lipoproteins. Due to its inherent structural dynamics and the presence of lipids, the structure of the biologically active apoE remains so far poorly described. To address this issue, we developed an innovative hybrid method combining experimental data with molecular modeling and dynamics to generate comprehensive models of the lipidated apoE4 isoform. Chemical cross-linking combined with mass spectrometry provided distance restraints, characterizing the three-dimensional organization of apoE4 molecules at the surface of lipidic nanoparticles. The ensemble of spatial restraints was then rationalized in an original molecular modeling approach to generate monomeric models of apoE4 that advocated the existence of two alternative conformations. These two models point towards an activation mechanism of apoE4 relying on a regulation of the accessibility of its receptor binding region. Further, molecular dynamics simulations of the dimerized and lipidated apoE4 monomeric conformations revealed an elongation of the apoE N-terminal domain, whereby helix 4 is rearranged, together with Arg172, into a proper orientation essential for lipoprotein receptor association. Overall, our results show how apoE4 adapts its conformation for the recognition of the low density lipoprotein receptor and we propose a novel mechanism of activation for apoE4 that is based on accessibility and remodeling of the receptor binding region.</p></div