Molecular Insights into Human Serum Albumin as ... - ACS Publications

Article Cite This: J. Am. Chem. Soc. 2017, 139, 15437-15445

pubs.acs.org/JACS

Molecular Insights into Human Serum Albumin as a Receptor of Amyloid-β in the Extracellular Region Tae Su Choi,†,‡,⊥ Hyuck Jin Lee,§,⊥ Jong Yoon Han,† Mi Hee Lim,*,∥ and Hugh I. Kim*,† †

Department of Chemistry, Korea University, Seoul 02841, Republic of Korea Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea § School of Life Sciences and ∥Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea Downloaded via FORDHAM UNIV on June 29, 2018 at 19:04:09 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Regulation of amyloid-β (Aβ) aggregation by metal ions and proteins is essential for understanding the pathology of Alzheimer’s disease (AD). Human serum albumin (HSA), a regulator of metal and protein transportation, can modulate metal−Aβ interactions and Aβ aggregation in human fluid; however, the molecular mechanisms for such activities remain unclear. Herein, we report the molecular-level complexation between Zn(II), Cu(II), Aβ, and HSA, which is able to alter the aggregation and cytotoxicity of Aβ peptides and induce their cellular transportation. In addition, a single Aβ monomerbound HSA is observed with the structural change of Aβ from a random coil to an α-helix. Small-angle X-ray scattering (SAXS) studies indicate that Aβ−HSA complexation causes no structural variation of HSA in solution. Conversely, ion mobility mass spectrometry (IM-MS) results present that Aβ prevents the shrinkage of the V-shaped groove of HSA in the gas phase. Consequently, for the first time, HSA is demonstrated to predominantly capture a single Aβ monomer at the groove using the phase transfer of a protein heterodimer from solution to the gas phase. Moreover, HSA sequesters Zn(II) and Cu(II) from Aβ while maintaining Aβ−HSA interaction. Therefore, HSA is capable of controlling metal-free and metal-bound Aβ aggregation and aiding the cellular transportation of Aβ via Aβ−HSA complexation. The overall results and observations regarding HSA, Aβ, and metal ions advance our knowledge of how protein− protein interactions associated with Aβ and metal ions could be linked to AD pathogenesis.

■

or small molecules targeting Aβ.16−25 An ideal strategy for inhibiting deleterious Aβ aggregation in the CNS, however, is still being developed, based on the fundamental understanding of Aβ-associated AD pathology.14,26 Notably, Aβ aggregation is observed exclusively in the CNS, but most nucleated cells in the human body secrete Aβ.15,27 Human serum albumin (HSA) (Figure 1b) is the most abundant plasma protein (ca. 0.4−0.6 mM), and more than ca. 90% of Aβ is expected to be bound to HSA in human blood plasma.28−33 Because of this direct interaction between Aβ and HSA, HSA is suggested as a key protein that possibly suppresses Aβ aggregation in the human body and reduces the risk of AD onset and progression.10,31,32,34−42 HSA not only sequesters most plasma Aβ from the aggregation-prone state31 but also assists the transportation of Aβ from the CSF to the plasma through the equilibrium shift.10 The plasma concentration of Aβ−HSA complexes decreases upon AD progression, which indicates that the equilibrium shift of Aβ mediated by HSA operates abnormally in the brains of AD patients.3

INTRODUCTION The etiology of Alzheimer’s disease (AD), the most prevalent neurodegenerative disease, has been suggested to be correlated with the aggregation of amyloid-β (Aβ) peptides in the central nervous system (CNS).1 Aβ peptides are the products from proteolytic cleavage of amyloid precursor protein (APP) by βand γ-secretases.2 Aβ peptides with 40 and 42 amino acid residues, Aβ40 and Aβ42 (Figure 1a), are the two most common isoforms of Aβ found in human cerebrospinal fluid (CSF).3 The aggregates of both isoforms, amyloid plaques, with various biological components, including metal ions and other proteins, are observed in the postmortem brain tissue of ADaffected patients.4 The formation of Aβ aggregates may be related to failure to clear Aβ from the human CSF5 and interaction with the lipid bilayer in cell membranes.6,7 The imbalance between the secretion and clearance rates of Aβ increases the concentration of Aβ in the CSF, thereby promoting the formation of cytotoxic Aβ aggregates (e.g., lipid-mediated aggregation) through variable pathways.2,8 Current approaches to prevent Aβ aggregation have focused on regulating Aβ levels in the extracellular region9−15 and blocking the aggregation of Aβ peptides by synthetic receptors © 2017 American Chemical Society

Received: August 12, 2017 Published: September 20, 2017 15437

DOI: 10.1021/jacs.7b08584 J. Am. Chem. Soc. 2017, 139, 15437−15445

Article

Journal of the American Chemical Society

Figure 1. Influence of HSA on aggregation, cytotoxicity, and cellular transportation of Aβ. (a) Representative structures of Aβ40 and Aβ42 generated from molecular dynamics (MD) simulations. (b) Structure of HSA from MD simulations based on X-ray crystallography (PDB 1BJ5). (c) TEM images of Aβ40 and Aβ42 aggregates incubated with and without HSA. Conditions: [HSA] = 20 μM; [Aβ] = 20 μM; 37 °C; 24 h; constant agitation. Scale bar = 0.2 μm. (d) Viability of 5Y cells treated with samples of preincubated (8 h) Aβ with and without HSA followed by 24 h incubation. Cell viability (%) was determined by the MTT assay compared to that of cells treated with a volume of H2O equal to the amount of protein added. Error bars represent the standard error from four independent experiments (P < 0.05). Conditions: [HSA] = 10 μM; [Aβ] = 10 μM. (e and f) Cellular membrane permeability of Hylite Fluor 488-conjugated Aβ40 (HF488Aβ40) incubated with HSA. Transmission (left) and fluorescence (right) images of 5Y cells, obtained by TIRF after 1 and 5 h incubation of HF488Aβ40 (e) without and (f) with HSA. Conditions: [HSA] = 250 nM; [HF488Aβ40] = 250 nM. Scale bar = 20 μm.

Herein, we demonstrate that HSA is able to modify the aggregation and cytotoxicity associated with Aβ and induce the cellular transportation of Aβ species through possibly forming Aβ−HSA complexes. Specifically, a single Aβ monomer could bind to the V-shaped groove of HSA, triggering its structural change from a random coil to an α-helix. As observed by solution small-angle X-ray scattering (SAXS), the overall dimensions of Aβ−HSA complexes are not significantly different from those of HSA. Unlike the solution state, in gas-phase ion mobility mass spectrometry (IM-MS), the groove region of HSA is contracted upon phase transfer from solution to gas, whereas the presence of an Aβ monomer in the groove prevents groove shrinkage. This approach is the first example to suggest the position of the ligand protein in protein heterodimers undergoing phase transfer from solution to the gas phase. HSA also forms heterogeneous complexes with small Aβ oligomers (2−7-mers on average) as suggested in previous studies.28,30,32,34 Moreover, HSA is capable of chelating out Zn(II) and Cu(II) from Aβ monomers and oligomers while maintaining binding with Aβ species. Our observations imply that HSA, as a molecular receptor of Aβ and a metal chelator, aids in regulating actions of Aβ peptides, including their aggregation, cytotoxicity, and cellular transportation with and without metal ions. Overall, more detailed information on the molecular interactions between Aβ species and HSA, described in this work, provides a better understanding of protein− protein interactions relevant to Aβ species and metal ions, which are linked to AD pathogenesis.

Although the molecular driving force of this equilibrium shift is unclear, plasma exchange with Grifols solution composed of 20%10 and 5%43 (w/v) HSA has been clinically applied to AD patients to lower the Aβ level in the CSF. The concentration of HSA is dramatically reduced in the CSF (ca. 1−5 μM) because it does not freely penetrate the blood−brain barrier (BBB).3,36 Nevertheless, HSA is still the most abundant protein (ca. 66%) in the CSF.36 Therefore, HSA is expected to delay Aβ aggregation in the CSF potentially through a direct interaction;31 both Aβ monomers and aggregates are reported to directly interact with HSA.42 Studies on the molecular interaction between HSA and Aβ species, however, have focused on examining the formation of Aβ aggregates.28,30,32,34 For this reason, characterization of the Aβ species−HSA complexes at the molecular level will be crucial for understanding the roles of HSA in Aβ regulation, including Aβ aggregation. HSA is a natural chelator of Zn(II) and Cu(II),44 metal ions that are abundant in the extracellular region within the human brain.45,46 Zn(II) and Cu(II) are known to facilitate Aβ oligomerization in the extracellular region.46 Dyshomeostasis of Zn(II) and Cu(II) is often observed in AD, and these metal cations are found in amyloid plaques of AD patients at high concentrations (ca. 1.0 mM for Zn and ca. 0.4 mM for Cu).45,47 HSA can sequester metal ions from Aβ monomers due to its high binding affinity for these ions [Ka for HSA−Cu(II) ≈ 1012 M−1 and Ka for HSA−Zn(II) ≈ 107 M−1; Ka for Aβ−Cu(II) ≈ 1010 M−1 and Ka for Aβ−Zn(II) ≈ 105 M−1],44,47−49 but Cu(II) in preformed Aβ fibrils is known not to be transferred to HSA.50 Hence, HSA prevents Cu(II)-induced Aβ aggregation in early stages and reduces the cytotoxicity of Aβ aggregates and radical formation by Cu(II).29,51 The molecular interactions between Aβ species and HSA in the presence of Zn(II) and Cu(II), however, have still not been revealed.

■

RESULTS AND DISCUSSION Influence of HSA on Aggregation, Cytotoxicity, and Cellular Transportation of Aβ. First, we investigated the effect of HSA on Aβ aggregation through transmission electron microscopy (TEM), thioflavin-T (ThT) assay, and gel electrophoresis with Western blot. Both Aβ40 and Aβ42 15438


Article


Figure 2. Interaction between HSA and Aβ40 or Aβ42. (a) ESI-MS spectra of HSA without Aβ, with Aβ40 (Aβ40:HSA = 1:1), with Aβ42 (Aβ42:HSA = 1:1), and with both Aβ40 and Aβ42 (Aβ40:Aβ42:HSA = 1:1:1). Conditions: [HSA] = 5 μM; [Aβ] = 5 μM. (b) ESI-CID-MS spectra corresponding to each spectrum in panel a. Charge states of HSA and Aβ−HSA complexes are marked as numbers in the MS spectra. (c) CID energy profiles of the Aβ−HSA complexes. (d) Magnified HSA peak with +16 charge state and Aβ−HSA peaks with +17 charge state, before (top) and after (bottom) the CID process.

species incubated without HSA exhibited fibrillar aggregates in TEM images, while the addition of HSA suppressed the formation of Aβ40 and Aβ42 fibers (Figure 1c). The ThT assay showed that HSA could delay the aggregation of Aβ40 at the initial stage, but the aggregation of Aβ42 was less postponed (Figure S1). Nevertheless, the maximum ThT intensities at the final stage (plateau) indicated that the amount of β-sheet-rich Aβ40 and Aβ42 aggregates was slightly reduced upon the addition of HSA. The size distributions of Aβ species incubated for 24 h with and without HSA were investigated through gel electrophoresis. Various-sized Aβ or HSA species that differed from the control samples (i.e., samples of Aβ only and HSA only) were not detected; however, Aβ−HSA complexes were observed (Figure S2). In the case of Aβ40, the proteins visualized by both Coomassie blue staining and Western blotting with an anti-Aβ antibody (6E10) displayed clear bands at ca. 70 kDa, which could be complexes of HSA (66.5 kDa) and Aβ40 (4.3 kDa) (Figure S2b). For Aβ42, the bands representing potent Aβ42−HSA complexes were observed by Coomassie blue staining, while the protein bands might be overlapped with smeared bands from oligomeric Aβ42 species once they were blotted with 6E10 (Figure S2c). More detailed studies about the formation of Aβ−HSA complexes were performed through mass spectrometric (MS) analysis (vide infra). In contrast, when HSA was added to the preformed Aβ aggregates, no significant change was observed in results of the ThT assay and TEM (Figure S3). Therefore, HSA might prefer to interact with small-sized Aβ species rather than higher-order Aβ aggregates. In addition, the impact of HSA on the cytotoxicity of Aβ aggregates in SH-SY5Y (5Y) cells was examined. When Aβ aggregates, generated by preincubation for 8 h, were added to 5Y cells and then incubated for 24 h, cell viability was ca. 64% and 59% upon addition of Aβ40 and Aβ42 aggregates, respectively (Figure 1d), while cell viability increased upon addition of Aβ species pretreated with HSA to cells (ca. 86% for Aβ40 and ca. 93% for Aβ42). Thus, HSA could reduce the toxicity induced by Aβ aggregates.

Since HSA was reported to be internalized from the extracellular region to cells through FcRn-mediated endocytosis,52 we investigated the capability of HSA to transport Aβ species through the membrane of 5Y cells using total internal reflection fluorescence (TIRF) and confocal microscopy. Hylite Fluor 488-conjugated Aβ40 (HF488Aβ40) was introduced into the medium of 5Y cells with and without HSA. In the absence of HSA, enhanced fluorescence of HF488Aβ40 was observed at the borders of 5Y cells after 1 h incubation of HF488Aβ40, indicating that HF488Aβ40 may be attached on the cellular membrane (Figure 1e). Little increased fluorescence intensity was shown after 5 h of incubation. On the other hand, intense fluorescence of HF488Aβ40 was observed within 5Y cells upon treatment with HSA even after only 1 h incubation (Figure 1f), and this fluorescence intensity was strengthened upon longer (5 h) incubation in 5Y cells. The same trends were observed by confocal microscopy (Figure S4). The confocal microscopic investigations confirmed that bovine serum albumin (BSA) contained in inactivated fetal bovine serum (FBS; an important component of medium for cell culture) did not significantly influence the transportation of Aβ species by HSA. Overall, the observations from both microscopic studies suggested that HSA could be involved in the transportation of Aβ species through the cellular membrane. Direct Interaction between Aβ Monomer and HSA. To identify the molecular interaction between Aβ and HSA, we performed native electrospray ionization mass spectrometry (ESI-MS).53−55 The ESI-MS spectrum of HSA (5 μM) exhibited major peaks around m/z 3800−4400 with charge states from +15 to +18 (Figure 2a).56 In the case where Aβ40:HSA = 1:1, Aβ40−HSA complex peaks (red circles with black border) appeared with charge states from +15 to +20, and HSA-unbound Aβ40 monomers with +3 (m/z 1444) and +4 (m/z 1084) charge states (unbordered red circles) were also observed (Figure 2a). For Aβ42:HSA = 1:1, the charge states of the complex peaks (yellow circles with black border) were identical to those of the Aβ40:HSA = 1:1 complex peaks. Aβ42 monomers (unbordered yellow circles) were also observed with 15439


Article


Figure 3. Structural characterization of Aβ monomer−HSA complexes. (a) MREs of HSA and Aβ−HSA complexes obtained from CD. Differences in helix propensities are presented in the inset CD spectra. (b) Rg values of HSA and Aβ−HSA complexes measured from Guinier approximation in SAXS. (c) CCS distribution of HSA and Aβ−HSA complexes indicated from IM-MS. (d and e) Representative candidate structures of HSA and Aβ−HSA complexes in (d) solution and (e) gas phase, obtained from the structural information in panels a−c and MD simulations.

abundances of bound Aβ40 and Aβ42 in HSA (i.e., transferred from solution to the gas phase) can be deduced. The relative abundances of dissociated Aβ40 and Aβ42 were similar at the same CID energy, implying that the ratio of bound Aβ40 and Aβ42 in HSA is almost equal. The average m/z value of fully protonated HSA in +16 charge state is theoretically 4156.3, but the nonspecifically bound salts shifted the m/z value of HSA to 4177.4 (Δm/z = ca. 21) (Figure 2d). Considering the shift of m/z by salts, theoretical average m/z values of fully protonated Aβ40 monomer−HSA (m/z 4187.7) and Aβ42 monomer−HSA (m/z 4198.5) in +17 charge state were well-matched with the experimental m/z values (m/z 4191.4 for Aβ40−HSA and 4193.4 for Aβ42−HSA). We also observed that extra Aβ remained unbound and caused no further m/z shift in the complex peaks (Figures S6 and S7). These results clearly indicate that HSA interacts with a single Aβ monomer. After CID, the m/z values of HSA and Aβ−HSA complexes converged to the theoretical m/z value of HSA with +16 charge state by the dissociation of salts and Aβ40 and Aβ42 monomers (Figure 2d, bottom). Overall, our observations indicate that HSA forms a 1:1 stoichiometric complex with Aβ40 and Aβ42. Structural Characterization of Aβ Monomer−HSA Complexes. Circular dichroism (CD) spectroscopy, SAXS, and IM-MS were utilized to characterize the secondary and tertiary structures of Aβ monomer−HSA complexes. On the

+3 (m/z 1506) and +4 (m/z 1130) charge states. In the case where Aβ40:Aβ42:HSA = 1:1:1 (Figure 2a, bottom), unbound Aβ40 and Aβ42 were ca. 55% and 45% (area of Aβ peaks; red and yellow circles), respectively, without a significant change in the complex peaks. Low-energy collision-induced dissociation (CID) was performed to confirm that the peaks in Figure 2a were those for Aβ−HSA complex (Figure 2b). In the cases where Aβ40:HSA = 1:1 and Aβ42:HSA = 1:1, Aβ40 and Aβ42 monomers (Figure 2b; red and yellow circles) with +2 to +4 charge states (+1 charge state was also observed with low abundance; see Figure S5) were dissociated from the complex peaks, clearly indicating that HSA formed a complex with both Aβ40 and Aβ42. During CID, the charge states of Aβ−HSA complexes (+15 to +20) were reduced to +15 to +19 in Aβunbound HSA peaks (black border) by increasing the relative abundances of +15 and +16 charge states. Since the total sum of charge states was conserved before and after the CID process, we expect that as the charge state of dissociated Aβ monomers increases (e.g., +3 and +4 charge states), the charge state of Aβ−HSA complexes releasing Aβ monomers increases (e.g., +18 to +20 charge states). For Aβ40:Aβ42:HSA = 1:1:1, Aβ40 and Aβ42 concurrently dissociated from HSA (Figure 2b, bottom). The area ratio of dissociated Aβ40 and Aβ42 peaks to complex peaks was monitored as the CID energy increased (Figure 2c). Although CID energy does not indicate the binding affinity between Aβ and HSA in solution, the relative 15440


Article

Journal of the American Chemical Society basis of deconvolution of CD spectra (Figure S8) using the Ka of Aβ40−HSA complex (2.0 × 105 M−1),57 the α-helix feature at 208 and 222 nm in Aβ40 within the protein complexes was confirmed as previously reported (Figure S9a).57 Then the helix propensity of Aβ40 on HSA was calculated by comparing the mean residue ellipticity (MRE) of the complex to HSA (Figure 3a).58 The same procedure was applied to estimate the secondary structure of Aβ42 (Figure S9b). Helix propensities of unbound Aβ40 and Aβ42 were approximately 10% (Figure S9c), but they were changed to 55% (for Aβ40) and 31% (for Aβ42) in the complexes (Figure 3a). Guinier analysis of SAXS patterns was performed to obtain the radius of gyration (Rg) of HSA and Aβ−HSA complexes in solution (Figure S10). They exhibited similar Rg values (ca. 30 Å), indicating that Aβ−HSA complexation did not change the overall size (Figure 3b). Collision cross section (CCS) values (Figure 3c) of HSA and Aβ−HSA complexes with a +17 charge state in the gas phase were also determined by traveling wave ion mobility spectrometry (TWIMS) and drift tube ion mobility spectrometry (DTIMS) measurements (Figure S11).59 In contrast to the SAXS results, the obtained CCS value of HSA (40.5 nm2) was smaller than the measured values of Aβ40−HSA (ca. 43.4 nm2) and Aβ42−HSA (ca. 43.1 nm2). To obtain structural details matching the results in Figure 3a−c, molecular dynamics (MD) simulations were performed at various locations of Aβ in HSA (Figure S12). Briefly, theoretical scattering profiles of MD-simulated structures were compared with experimental scattering profiles of HSA and Aβ−HSA complexes by use of the program CRYSOL (Figure S13).61 Then the lowest χ2 structures matched to the results from CD spectra (helix propensity 40−60% for Aβ40 and 21− 41% for Aβ42) were selected as the representative structures in solution (Figure 3d and Figure S14). Gas-phase MD simulations of the representative structures were also performed to determine their theoretical collision cross section (CCStheo) values by projected superposition approximation in PSA-web server (Figure S14).62 The average CCS values of MD-simulated structures were well-matched to experimental (CCSexp) values (Figure 3e). The representative MD-simulated structure of HSA was globular in solution (Figure 3d). Although the protein structure in the gas phase was contracted, with disappearance of the groove between domains 1 and 3 (Figure 3e). The structures of Aβ40−HSA and Aβ42−HSA complexes, where Aβ40 and Aβ42 were inserted into the groove, were also globular in solution (Figure 3d). In the gas phase, however, the conformations of the complexes were less collapsed than the conformation of HSA because Aβ in the groove sterically disrupted the approach between domains 1 and 3 (Figure 3e). For this reason, CCStheo and CCSexp values of Aβ−HSA complexes were larger than those of HSA, suggesting that the Aβ monomer could be inserted into the groove of HSA. When we further examined other cases (e.g., externally bound Aβ monomer−HSA and candidate structures with the highest χ2 values), all of them were not concurrently matched to experimental results in Figure 3a−c (see details in Supporting Information). Hence, the HSA groove was found to be the most likely binding site for both Aβ40 and Aβ42 monomers. Driving Force of the Secondary Structural Transition from Random Coil to α-Helix. α-Helix structure of Aβ peptides is commonly induced under low relative permittivity (εr) that enhances the intramolecular hydrogen bonds (Hbonds) between peptide backbones.63 The HSA groove would

also provide a low-εr environment to Aβ by blocking contact with water molecules, as previously reported in the interior regions of globular proteins.64,65 Thus, the solvent-accessible surface areas (SASA) of Aβ as a random coil (Figure 1a) and of HSA-bound states as an α-helix (Figure 3d) were investigated. SASA values of Aβ were 46.2 (±3.1) nm2 for Aβ40 and 48.1 (±3.1) nm2 for Aβ42, but those of HSA-bound Aβ were reduced to 26.4 (±2.6) nm2 for Aβ40 and 29.8 (±1.8) nm2 for Aβ42 (Figure 4a). Additionally, the number of intramolecular

Figure 4. Driving force of HSA-mediated secondary structural transitions of Aβ40 and Aβ42. (a) SASA values of Aβ in unbound and bound states in the HSA groove. (b) Number of intramolecular hydrogen bonds of Aβ corresponding to each state in panel a.

H-bonds within Aβ40 and Aβ42 increased from 10.7 (±3.1) and 10.0 (±3.2) to 20.0 (±3.5) and 20.2 (±1.5), respectively, when they were bound to HSA (Figure 4b). These results imply that the groove of HSA can block the contact of Aβ with water molecules, and thereby HSA-bound Aβ can form more intramolecular H-bonds than HSA-unbound Aβ under low εr. Vivekanandan et al. reported that a 310-helix structure is formed in the central hydrophobic region (His13−Asp23) of Aβ40.68 In our MD-simulated Aβ40 and Aβ42, α-helix residues with a few 310-helix residues (16% for Aβ40 and 26% for Aβ42) existed between His13 and the C-terminus (Figure S15), corresponding to the hydrophobic core region of Aβ fibril backbone.66,67 Compared to the 310-helix structures, the broader core region of Aβ (Val24 to C-terminus) is stabilized in helical conformation because the groove of HSA can protect the core region from contact with water molecules. Then the molecular interaction with HSA rearranges the structures of Aβ, mainly, to the α-helix form. In the case of Aβ42, its two hydrophobic C-terminal residues (Ile41 and Ala42) are known to enhance intermolecular interactions.69 Thus, the C-terminal region of Aβ42 destabilizes the intramolecular interaction within Aβ42 through direct interaction with HSA, thereby reducing the helix propensities in Aβ42. Nevertheless, SASA values and the number of H-bonds of Aβ42 were similar to those of Aβ40, implying that hydrophobic interactions and Hbonds promote the insertion of Aβ42 into the groove; therefore, the relative abundances of Aβ40 and Aβ42 bound to HSA were noted to be almost equal in CID experiments. Complex Formation between Heterogeneous Aβ Oligomers and HSA. In addition to Aβ monomers, it has been reported that the interaction between Aβ oligomers and HSA could modulate Aβ fibrillation.28,30,32 Thus their direct 15441


Article


Figure 5. Effects of HSA on aggregation, cytotoxicity, and cellular transportation of metal−Aβ species. (a) Proposed binding sites of Zn(II) and Cu(II) in Aβ at a physiologically relevant pH. (b) Previously reported binding sites of Zn(II) and Cu(II) in HSA. (c and g) TEM images of (c) Zn(II)- and (g) Cu(II)-mediated Aβ40/Aβ42 aggregates with and without HSA. Conditions: [HSA] = 20 μM; [Aβ] = 20 μM; [ZnCl2] or [CuCl2] = 20 μM; 37 °C; 24 h; constant agitation. Scale bar = 0.2 μm. (d and h) Viability of 5Y cells treated with samples generated upon 8 h preincubation of (d) Zn(II) or (h) Cu(II) with Aβ and HSA. After an additional 24 h of incubation with cells, cell viability (%) was determined by the MTT assay compared to cells treated with a volume of H2O equal to the protein added. Error bars represent the standard error from four independent experiments (P < 0.05). Conditions: [HSA] = 10 μM; [Aβ] = 10 μM; and [ZnCl2] or [CuCl2] = 10 μM. (e and f) Cellular transportation of Zn(II)treated Hylite Fluor 488-conjugated Aβ40 (HF488Aβ40) upon incubation with and without HSA. Transmission (left) and fluorescence (right) images of 5Y cells obtained by TIRF after 1 and 5 h incubation of HF488Aβ40 and Zn(II) with and without HSA are shown. Conditions: [HSA] = 250 nM; [HF488Aβ40] = 250 nM; and [ZnCl2] = 250 nM. Scale bar = 20 μm.

images showed that HSA induced the formation of smaller Aβ aggregates compared to aggregates that formed without HSA. HSA could also delay the kinetics of Zn(II)-associated Aβ40 aggregation and generate fewer β-sheet-rich Zn(II)−Aβ42 aggregates, as observed by the ThT assay (Figure S18); however, HSA could not disaggregate or modulate further aggregation of preformed Zn(II)-induced fibrillar aggregates of both Aβ40 and Aβ42 (Figure S19c, top row). In addition, clear bands at ca. 70 kDa, which could be protein complexes of HSA and Aβ40 or Aβ42 with Zn(II), were observed through gel electrophoresis visualized by Coomassie blue staining and Western blotting with an anti-Aβ antibody (6E10), similar to metal-free conditions (Figure S20b,c). On the basis of our observation, HSA preferably interacts with small Zn(II)−Aβ aggregates rather than higher-order Zn(II)−Aβ species, which could be associated with its ability to modulate Zn(II)−Aβ aggregation. The influence of HSA on toxicity trigged by Zn(II)−Aβ aggregates and their transportation through the cellular membrane was further investigated. Zn(II)−Aβ aggregates were cytotoxic in 5Y cells, resulting in ca. 63% and 51% cell viability for Zn(II)−Aβ40 and Zn(II)−Aβ42 aggregates, respectively (Figure 5d). In contrast, addition of HSA increased the viability of 5Y cells to ca. 84% and 74% upon exposure to Aβ40 and Aβ42 in the presence of Zn(II), respectively (Figure 5d). Moreover, the fluorescence of HF488Aβ with Zn(II) without HSA was noticeably observed in 5Y cells after 5 h, while intense fluorescence intensity of HF488Aβ with Zn(II) was detected in the presence of HSA even after 1 h by TIRF (Figure 5e,f), indicating that HSA could mediate the transportation of Aβ through the cellular membrane of 5Y cells in the presence of Zn(II). The same trends were observed in both FBS-treated and untreated cells by measuring fluorescence via confocal

interaction was investigated by native ESI-MS. Aβ was incubated without HSA for 24 h to convert monomers into various types of aggregates (from dimers to fibrils), followed by the addition of HSA. ESI-MS spectra of the mixture of HSA and the incubated Aβ aggregates exhibited m/z shifts and broadened major peak shapes compared to the spectrum of HSA alone (Figure S16a). The m/z distributions in Aβ40− HSA complexes corresponded to 6−7-mers of Aβ40 on average (Table S1), while those of Aβ42−HSA complexes corresponded to dimeric Aβ42 (Table S2). When Aβ40 and Aβ42 were incubated for 2 h prior to HSA treatment, the major peaks of Aβ-bound HSA were highly polydisperse because of the interaction with various-sized Aβ oligomers (Figure S16b). Previously, Melacini and co-workers reported that Aβ oligomers interact on the external surface of HSA domains.28,34 Since the dimension of the groove is similar to that of the Aβ monomer, Aβ oligomers may not be inserted into the groove due to their large sizes. Nevertheless, it is predicted that the interactions of HSA with oligomeric Aβ species might reduce the cytotoxicity induced by Aβ aggregates, as previously reported,42 and enhance the transportation of Aβ through the cellular membrane. Effects of HSA on Aggregation, Cytotoxicity, and Cellular Membrane Transportation of Aβ in the Presence of Metal Ions. Zn(II) and Cu(II) can be coordinated within Aβ (Figure S17) through interaction with amino acid residues located at the N-terminal region (Figure 5a),49,70 promoting the formation of cytotoxic Aβ aggregates.44,45,71,72 Since HSA was previously reported to disturb metal−Aβ interactions by binding metal ions at the N-terminal metal-binding site (NTS) or multiple metal-binding site (MBS; Figure 5b),50 the influence of HSA on metal-induced Aβ aggregation was examined. As depicted in Figure 5c, TEM 15442


Article


Figure 6. Transfer of metal ions from Aβ monomers and oligomers to HSA. (a) Magnified HSA peak with +16 charge state after CID of Aβ40 monomer−HSA and Aβ42 monomer−HSA. (b) Dissociated Aβ peaks with +4 charge state after CID process. (c) Comparison of CID energy profiles between Aβ−HSA−metal complexes and Aβ−HSA complexes. (d) Magnified ESI-CID-MS spectra of incubated (for 24 h) Aβ40−HSA and Aβ42−HSA complexes with and without metal ions.

Figure 7. Potent actions of HSA toward activities of Aβ species in the extracellular region. (a) Suppression and delay of Aβ aggregation by HSA. (b) HSA-mediated cellular transportation of Aβ monomers and oligomers. (c) Formation of Aβ monomer−HSA complex within the HSA groove. (d) Transfer of Zn(II) and Cu(II) from Aβ to HSA.

Considering the charge state of HSA (+16), the difference in the m/z values (Δm/z = ca. 4) could originate from the binding of Zn(II) (65.4 Da) and Cu(II) (63.5 Da). In addition, the dissociated Aβ40 and Aβ42 did not contain Zn(II) and Cu(II) (Figure 6b), implying that Zn(II) and Cu(II) might be transferred to HSA. Our structural analysis using SAXS, CD, and IM-MS showed that the structural characteristics of Aβ−HSA complexes were preserved regardless of the addition of Zn(II) and Cu(II) (Figures S10, S23, and S24). Furthermore, the CID energy profiles of Aβ−HSA complexes in the presence of Zn(II) and Cu(II) were similar to those without Zn(II) and Cu(II) (Figure 6c). These results clearly indicate that Zn(II) and Cu(II) were chelated by HSA independently of the interaction between HSA and Aβ monomers. Since the binding sites of Zn(II) (MBS) and Cu(II) (NTS) are located in different regions from major Aβ binding sites of HSA, as presented in Figure 5b, Aβ− HSA complex formation might not be disrupted by coordination of Zn(II) and Cu(II) in HSA. The transfer of Zn(II) and Cu(II) from small Aβ oligomers to HSA was also investigated. HSA formed a complex with Zn(II)- and Cu(II)induced small Aβ oligomers (Figure S25). As observed by ESICID-MS, HSA was found at m/z 4165 as a single Zn(II) or Cu(II) attached form (Figure 6d). Therefore, HSA could chelate out Zn(II) and Cu(II) from Aβ oligomers.

microscopy (Figure S21). Overall, HSA suppressed the cytotoxicity induced by Zn(II)−Aβ aggregates and supported their cellular transportation, as observed under metal-free conditions. HSA could modulate the aggregation of Cu(II)−Aβ (Figure 5g); however, preformed Cu(II)−Aβ aggregates might not be disassembled by incubation with HSA (Figure S19c, bottom row). Amorphous or smaller aggregates of Cu(II)−Aβ aggregates were monitored by TEM upon treatment with HSA (Figure 5g). Similar to metal-free and Zn(II)-present conditions, based on gel electrophoresis, protein bands at ca. 70 kDa corresponding to Aβ40/Aβ42−HSA complexes were detected in the presence of Cu(II) (Figure S20b,c). These results suggest that HSA could interact with Cu(II)−Aβ species. Furthermore, HSA also decreased the cytotoxicity triggered by Cu(II) and Aβ [Figure 5h; cell survival for Cu(II)−Aβ40 (from ca. 61% to 92%) and Cu(II)−Aβ42 (from ca. 46% to 63%)]. Taken together, the actions of HSA toward Aβ species upon treatment of Zn(II) and Cu(II) were similar to those in the absence of metal ions. Regardless of the absence and presence of metal ions, HSA could (i) regulate the aggregation pathways of Aβ and reduce their toxicity and (ii) affect cellular transportation of Aβ, potentially through the formation of Aβ−HSA complexes. Investigation of Aβ−HSA Complexes in the Presence of Zn(II) and Cu(II). Native ESI-MS revealed that both metalassociated monomeric Aβ40 and Aβ42 also formed complexes with HSA (Figure S22a). Low-energy CID of Aβ−HSA complexes with Zn(II) and Cu(II) resulted in dissociation of Aβ monomers from the complex ions. After CID, HSA ions with a +16 charge state were found at ca. m/z 4165 (Figure 6a), whereas the protonated m/z value with the same charge state was ca. 4161 (Figure 2) in the absence of metal ions.

■

CONCLUSION We characterized the molecular interaction between Zn(II), Cu(II), Aβ, and HSA to identify the regulatory activities of HSA in formation of Aβ aggregates (Figure 7a) and HSAmediated cellular transportation of Aβ species (Figure 7b) potentially by forming Aβ−HSA complexes with monomeric and oligomeric Aβ species. Moreover, using the phase-transfer 15443



■

strategy of complexes based on solution SAXS, IM-MS, and computational modeling, we demonstrate, for the first time, that HSA could (i) capture an Aβ monomer within its groove (Figure 7c) and (ii) serve as a metal chelator that prevents the interaction of Aβ with Zn(II) or Cu(II) (Figure 7d). These features of HSA, to the best of our knowledge, are likely the only system naturally occurring in the human body with high probability, given the large amount of HSA that exists in human fluid. In addition, since Aβ is secreted extracellularly as a monomeric form, the groove region of HSA will play a pivotal role as a molecular receptor of Aβ monomer. Thus, the molecular behavior of HSA toward Aβ monomer could be crucial for regulating the roles of Aβ in AD pathology. Furthermore, the molecular insights proposed herein can advance our understanding of protein−protein interactions associated with Aβ and metal ions, along with their influence on AD pathogenesis.

■

REFERENCES

(1) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353−356. (2) Haass, C.; Selkoe, D. J. Nat. Rev. Mol. Cell Biol. 2007, 8, 101−112. (3) Yamamoto, K.; Shimada, H.; Koh, H.; Ataka, S.; Miki, T. Geriatr. Gerontol. Int. 2014, 14, 716−723. (4) Iwatsubo, T.; Odaka, A.; Suzuki, N.; Mizusawa, H.; Nukina, N.; Ihara, Y. Neuron 1994, 13, 45−53. (5) Mawuenyega, K. G.; Sigurdson, W.; Ovod, V.; Munsell, L.; Kasten, T.; Morris, J. C.; Yarasheski, K. E.; Bateman, R. J. Science 2010, 330, 1774. (6) Di Scala, C.; Yahi, N.; Boutemeur, S.; Flores, A.; Rodriguez, L.; Chahinian, H.; Fantini, J. Sci. Rep. 2016, 6, 28781. (7) Kotler, S. A.; Walsh, P.; Brender, J. R.; Ramamoorthy, A. Chem. Soc. Rev. 2014, 43, 6692−6700. (8) Korshavn, K. J.; Bhunia, A.; Lim, M. H.; Ramamoorthy, A. Chem. Commun. 2016, 52, 882−885. (9) Sevigny, J.; Chiao, P.; Bussière, T.; Weinreb, P. H.; Williams, L.; Maier, M.; Dunstan, R.; Salloway, S.; Chen, T.; Ling, Y.; O’Gorman, J.; Qian, F.; Arastu, M.; Li, M.; Chollate, S.; Brennan, M. S.; QuinteroMonzon, O.; Scannevin, R. H.; Arnold, H. M.; Engber, T.; Rhodes, K.; Ferrero, J.; Hang, Y.; Mikulskis, A.; Grimm, J.; Hock, C.; Nitsch, R. M.; Sandrock, A. Nature 2016, 537, 50−56. (10) Boada, M.; Ortiz, P.; Anaya, F.; Hernández, I.; Munoz, J.; Nunez, L.; Olazaran, J.; Roca, I.; Cuberas, G.; Tarraga, L.; Buendia, M.; Pla, R. P.; Ferrer, I.; Paez, A. Drug News Perspect. 2009, 22, 325−339. (11) Doody, R. S.; Thomas, R. G.; Farlow, M.; Iwatsubo, T.; Vellas, B.; Joffe, S.; Kieburtz, K.; Raman, R.; Sun, X.; Aisen, P. S.; Siemers, E.; Liu-Seifert, H.; Mohs, R. N. Engl. J. Med. 2014, 370, 311−321. (12) Salloway, S.; Sperling, R.; Fox, N. C.; Blennow, K.; Klunk, W.; Raskind, M.; Sabbagh, M.; Honig, L. S.; Porsteinsson, A. P.; Ferris, S.; Reichert, M.; Ketter, N.; Nejadnik, B.; Guenzler, V.; Miloslavsky, M.; Wang, D.; Lu, Y.; Lull, J.; Tudor, I. C.; Liu, E.; Grundman, M.; Yuen, E.; Black, R.; Brashear, H. R. N. Engl. J. Med. 2014, 370, 322−333. (13) Derrick, J. S.; Lee, J.; Lee, S. J. C.; Kim, Y.; Nam, E.; Tak, H.; Kang, J.; Lee, M.; Kim, S. H.; Park, K.; Cho, J.; Lim, M. H. J. Am. Chem. Soc. 2017, 139, 2234−2244. (14) Selkoe, D. J. Ann. Neurol. 2013, 74, 328−336. (15) Bates, K. A.; Verdile, G.; Li, Q. X.; Ames, D.; Hudson, P.; Masters, C. L.; Martins, R. N. Mol. Psychiatry 2009, 14, 469−486. (16) Sinha, S.; Lopes, D. H. J.; Du, Z. M.; Pang, E. S.; Shanmugam, A.; Lomakin, A.; Talbiersky, P.; Tennstaedt, A.; McDaniel, K.; Bakshi, R.; Kuo, P. Y.; Ehrmann, M.; Benedek, G. B.; Loo, J. A.; Klarner, F.-G.; Schrader, T.; Wang, C. Y.; Bitan, G. J. Am. Chem. Soc. 2011, 133, 16958−16969. (17) Schrader, T.; Bitan, G.; Klarner, F.-G. Chem. Commun. 2016, 52, 11318−11334. (18) Lee, H. H.; Choi, T. S.; Lee, S. J. C.; Lee, J. W.; Park, J.; Ko, Y. H.; Kim, W. J.; Kim, K. M.; Kim, H. I. Angew. Chem., Int. Ed. 2014, 53, 7461−7465. (19) Kumar, S.; Hamilton, A. D. J. Am. Chem. Soc. 2017, 139, 5744− 5755. (20) Beck, M. W.; Derrick, J. S.; Kerr, R. A.; Oh, S. B.; Cho, W. J.; Lee, S. J. C.; Ji, Y.; Han, J.; Tehrani, Z. A.; Suh, N.; Kim, S.; Larsen, S. D.; Kim, K. S.; Lee, J.-Y.; Ruotolo, B. T.; Lim, M. H. Nat. Commun. 2016, 7, 13115. (21) Lee, S. J. C.; Nam, E.; Lee, H. J.; Savelieff, M. G.; Lim, M. H. Chem. Soc. Rev. 2017, 46, 310−323. (22) Derrick, J. S.; Kerr, R. A.; Nam, Y.; Oh, S. B.; Lee, H. J.; Earnest, K. G.; Suh, N.; Peck, K. L.; Ozbil, M.; Korshavn, K. J.; Ramamoorthy, A.; Prabhakar, R.; Merino, E. J.; Shearer, J.; Lee, J.-Y.; Ruotolo, B. T.; Lim, M. H. J. Am. Chem. Soc. 2015, 137, 14785−14797. (23) Lee, H. J.; Korshavn, K. J.; Nam, Y.; Kang, J.; Paul, T. J.; Kerr, R. A.; Youn, I. S.; Ozbil, M.; Kim, K. S.; Ruotolo, B. T.; Prabhakar, R.; Ramamoorthy, A.; Lim, M. H. Chem. Eur. J. 2017, 23, 2706−2715. (24) Beck, M. W.; Oh, S. B.; Kerr, R. A.; Lee, H. J.; Kim, S. H.; Kim, S.; Jang, M.; Ruotolo, B. T.; Lee, J.-Y.; Lim, M. H. Chem. Sci. 2015, 6, 1879−1886.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b08584. Additional text with Materials and Methods section and additional examination of MD-simulated structures; five tables listing theoretical helix propensities, m/z values, and CCS values; and 27 figures as described in the text (PDF)

■

Article

AUTHOR INFORMATION

Corresponding Authors

*[email protected] (M.H.L.) *[email protected] (H.I.K.) ORCID

Mi Hee Lim: 0000-0003-3377-4996 Author Contributions ⊥

T.S.C. and H.J.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by funding from the National Research Foundation of Korea (NRF), funded by the Korean government [NRF-2016R1A2B4013089 and 20100020209 (to H.I.K.); NRF-2017R1A2B3002585 and NRF2016R1A5A1009405 (to M.H.L.)]; the Korea University Future Research Grant (to H.I.K.); and the Nine Bridges Program Research Fund (Project 1.170051.01) and the Futureleading Specialized Research Fund (Project 1.170009.01) of UNIST (to M.H.L.). T.S.C. acknowledges the support from a T.J. Park Fellowship and Korea University Grant. We acknowledge Agilent Technologies Inc. for 6560 LC-IMS QTOFMS instrument support and technical/scientific advice. The synchrotron X-ray scattering measurements at 4C SAXS II beamline of the Pohang Accelerator Laboratory were supported by the Ministry of Education and Science Technology.73 This work was also supported by the National Institute of Supercomputing and Network/Korea Institute of Science and Technology Information with supercomputing resources including technical support (KSC-2016-C2-0021). We thank Dr. Yeon Ju Kwak for assistance with initial cell viability measurements. 15444


Article

Journal of the American Chemical Society (25) Malishev, R.; Nandi, S.; Kolusheva, S.; Levi-Kalisman, Y.; Klärner, F.-G.; Schrader, T.; Bitan, G.; Jelinek, R. ACS Chem. Neurosci. 2015, 6, 1860−1869. (26) Graham, W. V.; Bonito-Oliva, A.; Sakmar, T. P. Annu. Rev. Med. 2017, 68, 413−430. (27) Haass, C.; Selkoe, D. J. Cell 1993, 75, 1039−1042. (28) Algamal, M.; Milojevic, J.; Jafari, N.; Zhang, W.; Melacini, G. Biophys. J. 2013, 105, 1700−1709. (29) Perrone, L.; Mothes, E.; Vignes, M.; Mockel, A.; Figueroa, C.; Miquel, M. C.; Maddelein, M. L.; Faller, P. ChemBioChem 2010, 11, 110−118. (30) Milojevic, J.; Melacini, G. Biophys. J. 2011, 100, 183−192. (31) Stanyon, H. F.; Viles, J. H. J. Biol. Chem. 2012, 287, 28163− 28168. (32) Milojevic, J.; Raditsis, A.; Melacini, G. Biophys. J. 2009, 97, 2585−2594. (33) Kuo, Y.-M.; Kokjohn, T. A.; Kalback, W.; Luehrs, D.; Galasko, D. R.; Chevallier, N.; Koo, E. H.; Emmerling, M. R.; Roher, A. E. Biochem. Biophys. Res. Commun. 2000, 268, 750−756. (34) Milojevic, J.; Esposito, V.; Das, R.; Melacini, G. J. Am. Chem. Soc. 2007, 129, 4282−4290. (35) Milojevic, J.; Costa, M.; Ortiz, A. M.; Jorquera, J. I.; Melacini, G. J. Alzheimers Dis. 2014, 38, 753−765. (36) Bohrmann, B.; Tjernberg, L.; Kuner, P.; Poli, S.; Levet-Trafit, B.; Näslund, J.; Richards, G.; Huber, W.; Döbeli, H.; Nordstedt, C. J. Biol. Chem. 1999, 274, 15990−15995. (37) Dominguez-Prieto, M.; Velasco, A.; Vega, L.; Tabernero, A.; Medina, J. M. J. Alzheimers Dis. 2017, 55, 171−182. (38) Ezra, A.; Rabinovich-Nikitin, I.; Rabinovich-Toidman, P.; Solomon, B. J. Alzheimers Dis. 2016, 50, 175−188. (39) Wallin, C.; Luo, J.; Jarvet, J.; Wärmländer, S. K. T. S.; Gräslund, A. Isr. J. Chem. 2017, 57, 674−685. (40) Liu, Y.-H.; Wang, Y.-R.; Xiang, Y.; Zhou, H.-D.; Giunta, B.; Mañucat-Tan, N. B.; Tan, J.; Zhou, X.-F.; Wang, Y.-J. Mol. Neurobiol. 2015, 51, 1−7. (41) Han, S.-H.; Park, J.-C.; Mook-Jung, I. Prog. Neurobiol. 2016, 137, 17−38. (42) Wang, C.; Cheng, F.; Xu, L.; Jia, L. RSC Adv. 2016, 6, 71165− 71175. (43) Boada, M.; Anaya, F.; Ortiz, P.; Olazaran, J.; Shua-Haim, J. R.; Obisesan, T. O.; Hernandez, I.; Munoz, J.; Buendia, M.; Alegret, M.; Lafuente, A.; Tarraga, L.; Nunez, L.; Torres, M.; Grifols, J. R.; Ferrer, I.; Lopez, O. L.; Paez, A. J. Alzheimers Dis. 2017, 56, 129−143. (44) Bal, W.; Sokolowska, M.; Kurowska, E.; Faller, P. Biochim. Biophys. Acta 2013, 1830, 5444−5455. (45) Lee, H. J.; Korshavn, K. J.; Kochi, A.; Derrick, J. S.; Lim, M. H. Chem. Soc. Rev. 2014, 43, 6672−6682. (46) Leal, S. S.; Botelho, H. M.; Gomes, C. M. Coord. Chem. Rev. 2012, 256, 2253−2270. (47) Faller, P.; Hureau, C.; Berthoumieu, O. Inorg. Chem. 2013, 52, 12193−12206. (48) Xie, B.; Liu, F.; Dong, X.; Wang, Y.; Liu, X. M.; Sun, Y. J. Inorg. Biochem. 2017, 171, 67−75. (49) Faller, P.; Hureau, C.; La Penna, G. Acc. Chem. Res. 2014, 47, 2252−2259. (50) Jiang, D. L.; Zhang, L.; Grant, G. P. G.; Dudzik, C. G.; Chen, S.; Patel, S.; Hao, Y. Q.; Millhauser, G. L.; Zhou, F. M. Biochemistry 2013, 52, 547−556. (51) Lu, N.; Yang, Q.; Li, J.; Tian, R.; Peng, Y.-Y. Eur. J. Pharmacol. 2015, 767, 160−164. (52) Andersen, J. T.; Dalhus, B.; Cameron, J.; Daba, M. B.; Plumridge, A.; Evans, L.; Brennan, S. O.; Gunnarsen, K. S.; Bjørås, M.; Sleep, D.; Sandlie, I. Nat. Commun. 2012, 3, 610. (53) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1−23. (54) Göth, M.; Pagel, K. Anal. Bioanal. Chem. 2017, 409, 4305−4310. (55) Seo, J.; Hoffmann, W.; Warnke, S.; Huang, X.; Gewinner, S.; Schöllkopf, W.; Bowers, M. T.; von Helden, G.; Pagel, K. Nat. Chem. 2017, 9, 39−44.

(56) Bush, M. F.; Hall, Z.; Giles, K.; Hoyes, J.; Robinson, C. V.; Ruotolo, B. T. Anal. Chem. 2010, 82, 9557−9565. (57) Rózga, M.; Kłoniecki, M.; Jabłonowska, A.; Dadlez, M.; Bal, W. Biochem. Biophys. Res. Commun. 2007, 364, 714−718. (58) Chen, Y.-H.; Yang, J. T.; Chau, K. H. Biochemistry 1974, 13, 3350−3359. (59) Ruotolo, B. T.; Benesch, J. L. P.; Sandercock, A. M.; Hyung, S.J.; Robinson, C. V. Nat. Protoc. 2008, 3, 1139−1152. (60) Lee, J. W.; Kim, H. I. Analyst 2015, 140, 661−669. (61) Svergun, D.; Barberato, C.; Koch, M. H. J. J. Appl. Cryst. 1995, 28, 768−773. (62) Bleiholder, C.; Contreras, S.; Do, T. D.; Bowers, M. T. Int. J. Mass Spectrom. 2013, 345−347, 89−96. (63) Crescenzi, O.; Tomaselli, S.; Guerrini, R.; Salvadori, S.; D’Ursi, A. M.; Temussi, P. A.; Picone, D. Eur. J. Biochem. 2002, 269, 5642− 5648. (64) Dwyer, J. J.; Gittis, A. G.; Karp, D. A.; Lattman, E. E.; Spencer, D. S.; Stites, W. E.; García-Moreno E, B. Biophys. J. 2000, 79, 1610− 1620. (65) Sheinerman, F. B.; Honig, B. J. Mol. Biol. 2002, 318, 161−177. (66) Paravastu, A. K.; Leapman, R. D.; Yau, W.-M.; Tycko, R. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 18349−18354. (67) Wälti, M. A.; Ravotti, F.; Arai, H.; Glabe, C. G.; Wall, J. S.; Böckmann, A.; Güntert, P.; Meier, B. H.; Riek, R. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E4976−E4984. (68) Vivekanandan, S.; Brender, J. R.; Lee, S. Y.; Ramamoorthy, A. Biochem. Biophys. Res. Commun. 2011, 411, 312−316. (69) Lv, Z.; Roychaudhuri, R.; Condron, M. M.; Teplow, D. B.; Lyubchenko, Y. L. Sci. Rep. 2013, 3, 2880. (70) Alies, B.; Conte-Daban, A.; Sayen, S.; Collin, F.; Kieffer, I.; Guillon, E.; Faller, P.; Hureau, C. Inorg. Chem. 2016, 55, 10499− 10509. (71) Viles, J. H. Coord. Chem. Rev. 2012, 256, 2271−2284. (72) Tougu, V.; Tiiman, A.; Palumaa, P. Metallomics 2011, 3, 250− 261. (73) Kim, K.-W.; Kim, J.; Yun, Y. D.; Ahn, H.; Min, B.; Kim, N. H.; Rah, S.; Kim, H.-Y.; Lee, C.-S.; Seo, I. D.; Lee, W.-W.; Choi, H. J.; Jin, K. S. Biodesign 2017, 5, 24−29.

15445


Molecular Insights into Human Serum Albumin as ... - ACS Publications

Recommend Documents