Mellitate: A multivalent anion with extreme charge density causes rapid aggregation and misfolding of wild type lysozyme at neutral pH

<div><p>Due to its symmetric structure and abundance of carboxyl groups, mellitic acid (MA–benzenehexacarboxylic acid) has an uncommon capacity to form highly ordered molecular networks. Dissolved in water, MA dissociates to yield various mellitate anions with pronounced tendencies to form complexes with cations including protonated amines. Deprotonation of MA at physiological pH produces anions with high charge densities (MA^5- and MA^6-) whose influence on co-dissolved proteins has not been thoroughly studied. As electrostatic attraction between highly symmetric MA^6- anions and positively charged low-symmetry globular proteins could lead to interesting self-assembly patterns we have chosen hen egg white lysozyme (HEWL), a basic stably folded globular protein as a cationic partner for mellitate anions to form such hypothetical nanostructures. Indeed, mixing of neutral HEWL and MA solutions does result in precipitation of electrostatic complexes with the stoichiometry dependent on pH. We have studied the self-assembly of HEWL-MA structures using vibrational spectroscopy (infrared absorption and Raman scattering), circular dichroism (CD), atomic force microscopy (AFM). Possible HEWL-MA^6- molecular docking scenarios were analyzed using computational tools. Our results indicate that even at equimolar ratios (in respect to HEWL), MA^5- and MA^6- anions are capable of inducing misfolding and aggregation of the protein upon mild heating which results in non-native intermolecular beta-sheet appearing in the amide I’ region of the corresponding infrared spectra. The association process leads to aggregates with compacted morphologies entrapping mellitate anions. The capacity of extremely diluted mellitate anions (i.e. at sub-millimolar concentration range) to trigger aggregation of proteins is discussed in the context of mechanisms of misfolding.</p></div

Dependency of HEWL-MA complex stoichiometry on pD.

<p>Approximate stoichiometries of HEWL-MA complexes precipitating from D₂O-based mixtures of MA and HEWL (at the initial 21:1 molar ratio) at different pD (black squares connected with straight lines). Samples of freshly mixed MA and HEWL were subjected to 30 min incubation at 65°C and subsequently centrifuged; pellets were alkalized with a portion of diluted NaOD to pD = 12 resulting in solubilization of aggregates. Ratio of IR absorption at 1590 and 1650 cm^-1 was used to estimate MA:HEWL molar ratio in the precipitate. The overlaid plot (black dots) corresponds to “theoretical” stoichiometry obtained as a molar ratio of MA and HEWL ions required to give neutral net charge of the complex in the absence of other ions (based on pH-dependent protonation equilibria of both HEWL and MA uncorrected for isotopic effects).</p

Hypothetical docking interactions between HEWL and MA^5- and MA^6- anions.

<p>(A) Visualization of docking MA^5- anion on HEWL native monomer involving residues Lys116 and Arg112 (according to AutoDock). (B) Binding energies of MA^5- and MA^6- to three different clusters of positively charges amino acid residues on the native HEWL molecule: Cluster 1: Arg5, Lys33, Arg1250; Cluster 2: Arg45, Arg68; Cluster 3: Arg21, Arg112, and Lys116. (C) Primary structure of HEWL with aggregation-prone segments (according to TANGO) marked in yellow and the MA^5- / MA^6- –binding clusters (residues) in bold (number in superscript corresponds to the cluster number).</p

Co-precipitation of MA and HEWL.

<p>(A) Light scattering on HEWL-MA complexes formed at different molar ratios, HEWL wt. concentration was kept constant at 0,06%, pH was set at approx. 7,4; Inset shows magnification of the scattering dependency for the lowest MA:HEWL molar ratios; The photo shows 1 wt. %, pH 7,4 solutions of HEWL (left), MA (right) and their 1:1 mixture (middle); the molecular structure of MA is placed above the inset plot. (B) Infrared spectra of HEWL and MA dissolved in D₂O at pD approx. 4 and 8 (solvent subtracted).</p

AFM images of representative specimen of HEWL and HEWL-MA aggregates obtained through prolonged incubation at 65°C.

<p>AFM images of representative specimen of HEWL aggregates obtained through prolonged incubation in the absence (left column) or presence (center and right columns) of MA under different conditions (2 wt. % HEWL-MA complexes in D₂O under essentially the same conditions as used for the preparation of samples for the IR spectroscopic examination reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187328#pone.0187328.g002" target="_blank">Fig 2</a>). The scale bar corresponds to all images. Inset image corresponds to fresh room temperature precipitate from 21:1 MA:HEWL sample before incubation at 65°C.</p

Fluorescence emission spectra of ThT-stained aggregates obtained through mixing of MA and HEWL solutions in D₂O.

<p>Complexes were prepared by mixing samples at pD 7.6 and 21:1 (A) and 1:1 (B) MA:HEWL molar ratios. Prior to measurements, aggregates were incubated at 65^°C / 300 rpm for specified periods of time (RT denotes spectra of freshly prepared samples). Inset in panel B shows emission spectra of ThT-stained HEWL samples in the absence of MA. All spectra shown were collected for identical ThT and protein concentrations, and using identical optical pathways and photomultiplier gain (voltage) settings; λ_exc. = 440 nm.</p

Time-dependent MA-induced misfolding of HEWL at 65°C probed by FT-IR spectroscopy.

<p>FT-IR spectra were acquired in the absence (A) and presence (B-D) of MA at different MA:HEWL molar ratios: 1:1 (B), 5,25:1 (C), and 21:1 (D). Concentration of HEWL in all samples was kept constant at 2 wt. %, pD of HEWL and MA solutions prior to mixing was adjusted to 7,5. The D₂O-subtracted spectra correspond to pure precipitates (pellets) separated from the bulk sample on a centrifuge and re-suspended in D₂O.</p

MA-induced aggregation of HEWL traced by CD spectroscopy.

<p>Far-UV CD spectra of HEWL (A), and of HEWL in the presence of MA at 1:1 molar ratio (B) at increasing temperatures and after cooling to 25 ^oC, pD of samples was 7.4. Inset shows quantitative plots of ellipticity at 208 and 222 nm for either case.</p

On the Function and Fate of Chloride Ions in Amyloidogenic Self-Assembly of Insulin in an Acidic Environment: Salt-Induced Condensation of Fibrils

Formation of amyloid fibrils is often facilitated in the presence of specific charge-compensating ions. Dissolved sodium chloride is known to accelerate insulin fibrillation at low pH that has been attributed to the shielding of electrostatic repulsion between positively charged insulin molecules by chloride ions. However, the subsequent fate of Cl^– anions; that is, possible entrapment within elongating fibrils or escape into the bulk solvent, remains unclear. Here, we show that, while the presence of NaCl at the onset of insulin aggregation induces structural variants of amyloid with distinct fingerprint infrared features, a delayed addition of salt to fibrils that have been already formed in its absence and under quiescent conditions triggers a “condensation effect”: amyloid superstructures with strong chiroptical properties are formed. Chloride ions appear to stabilize these superstructures in a manner similar to stabilization of DNA condensates by polyvalent cations. The concentration of residual chloride ions trapped within bovine insulin fibrils grown in 0.1 M NaCl, at pD 1.9, and rinsed extensively with water afterward is less than 1 anion per 16 insulin monomers (as estimated using ion chromatography) implying absence of defined solvent-sequestered nesting sites for chloride counterions. Our results have been discussed in the context of mechanisms of insulin aggregation

Master and Slave Relationship Between Two Types of Self-Propagating Insulin Amyloid Fibrils

Cross-seeding of fibrils of bovine insulin (BI) and Lys^B31-Arg^B32 human insulin analog (KR) induces self-propagating amyloid variants with infrared features inherited from mother seeds. Here we report that when native insulin (BI or KR) is simultaneously seeded with mixture of equal amounts of both templates (i.e., of separately grown fibrils of BI and KR), the phenotype of resulting daughter fibrils is as in the case of the purely homologous seeding: heterologous cotemplates accelerate the fibrillation but do not determine infrared traits of the daughter amyloid. This implies that fibrillation-promoting and structure-imprinting properties of heterologous seeds become uncoupled in the presence of homologous seeds. We argue that explanation of such behavior requires that insulin molecules partly transformed through interactions with heterologous fibrils are subsequently recruited by homologous seeds. The selection bias toward homologous daughter amyloid is exceptional: more than 200-fold excess of heterologous seed is required to imprint its structural phenotype upon mixed seeding. Our study captures a snapshot of elusive docking interactions in statu nascendi of elongation of amyloid fibril and suggests that different types of seeds may collaborate in sequential processing of soluble protein into fibrils