Purified AMBN DelEx5 was incubated with 1,000 units of recombinant PKA Cα1 (New England Biolabs, Ipswich, MA) in PKA reaction buffer, or 5,000 units CK2 (New England Biolabs) in CK2 reaction buffer supplied with 0.1 μl γ-[32P] ATP (~6,000 Ci/mmol, PerkinElmer, Waltham, MA, USA) in a total volume of 20 μl for 30 min at 30°C. The reactions were terminated by addition of 4 μl SDS loading buffer and boiled for 10 min. Subsequently the samples were separated by SDS-PAGE in 12% Tris-HCl (BioRad, Hercules, CA, USA), and the bands were exposed by Coomassie stain and the radioactivity was detected by exposure to CL-Xposure film (ThermoFischer Scientific, Waltham, MA, USA).

His-tagged (1 μg) PKA Cα1 (Millipore, Billerica, MA, USA,) or his-tagged (1 μg) CK2 (ATGen Ltd, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea,) was incubated with purified AMBN-WT (100 pmol), C-terminus (314 pmol), N-terminus (100 pmol), or DelEx5 (100 pmol) in CK2 or PKA reaction buffer supplied with 1 mM ATP in a total volume of 20 μl at 30°C for 24 h. Ten picomoles from each of the samples were submitted to LC-ESI-MS analyses. The remaining of the samples were boiled in Laemmli buffer, loaded on a 12% Ready Gel Tris-HCL (BioRad), and separated by electrophoresis, and the gel stained for phospho-proteins with Pro-Q® Diamond Phosphoprotein Gel Stain and destained with Pro-Q® Diamond Phosphoprotein Gel Destaining Solution (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer's protocol. The phosphor luminiscent gel bands were then scanned at in ChemiDoc XRS+ imaging system (BioRad) at 510 nm.

To detect phosphorylation sites of AMBN, 10 pmol of protein was digested by adding 0.2 μg trypsin in 20 μl 25 mM ammonium bicarbonate and incubation for 16 h at 37°C. The digestion was stopped by adding 2 μl 5% formic acid. The generated peptides were purified using an OMIX C18 (Agilent, Santa Clara, CA, USA), and dried using a Speed Vac concentrator (Concentrator Plus, Eppendorf, Hamburg, Germany).

The tryptic peptides were dissolved in 10 μl 0.1% formic acid/2% acetonitrile and 5 μl analyzed using an Ultimate 3000 RSLCnano-UHPLC system connected to a Q Exactive mass spectrometer (ThermoFisher Scientific) equipped with a nano electrospray ion source. For liquid chromatography separation, an Acclaim PepMap 100 column (C18, 2 μm beads, 100 Å, 75 μm inner diameter, 50 cm length) (Dionex, Sunnyvale CA, USA) was used. A flow rate of 300 nL/min was employed with a solvent gradient of 4–35% B in 47 min, to 50% B in 10 min and then to 80% B in 3 min. Solvent A was 0.1% formic acid and solvent B was 0.1% formic acid/90% acetonitrile. The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 300 to 2,000) were acquired with the resolution R = 70,000 at m/z 200, after accumulation to a target of 1e6. The maximum allowed ion accumulation times were 60 ms. The method used allowed sequential isolation of up to the 10 most intense ions, depending on signal intensity (intensity threshold 1.7e4), for fragmentation using higher-energy collisional induced dissociation (HCD) at a target value of 10,000 charges and a resolution R = 17,500 Target ions already selected for MS/MS were dynamically excluded for 60 s. The isolation window was m/z = 2 without offset. For accurate mass measurements, the lock mass option was enabled in MS mode.

Data were acquired using Xcalibur v2.5.5 and raw files were processed to generate peak list in Mascot generic format (^*.mgf) using ProteoWizard release version 3.0.331. Database searches were performed using Mascot in-house version 2.4.0 to search the SwissProt database (Human, 20.279 proteins), assuming the digestion enzyme trypsin at maximum one cleavage site, fragment ion mass tolerance of 0.05 Da, parent ion tolerance of 10 ppm, and oxidation of methionines, and acetylation of the protein N-terminus as variable modifications. Scaffold (version Scaffold_4.4.8, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95.0% probability by the Scaffold Local FDR algorithm.

In dynamic light scattering (DLS), a particle in solution is illuminated by laser and scatter light that fluctuate with the random movement (Brownian motion) of the particles. As movement of a particle is inversely correlated with its mass, the light scattering fluctuations may be used to estimate particle size. DLS was measured in a Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK) in DTS1070 disposable foldable capillary cells (Malvern Instruments Ltd) at 12°C, using a backscatter angle of 173°, an equilibration time of 5 min and three replications per sample. Analysis was done in the Zetasizer Software v7.11 (Malvern Instruments Ltd). The dispersant was chosen in the software as water with a refractive index of 1.330 and viscosity 0.8872 cP, the particle protein with refractive index 1.450 and absorption 0.001. The distribution analysis was done using the protein analysis option in the software which is a non-negative least squares analysis with an automatically determined regularizer and L-curve analysis.

Small angle X-ray scattering is used to gather information about the structure of proteins in solution. This information is obtained by analyzing the intensity of photons (I) scattered in terms of the scattering vectors (s). To analyze the degree of folding of macromolecules, I(s)·(s)² against (s nm⁻¹) is plotted in a Kratky plot (Putnam et al., 2007). Folded macromolecules have a high I at lower values of (s), illustrated as a peak in the plot. For a disordered and elongated protein, the function of I is linear and reaches a plateau at higher (s) values. SAXS was performed at the European Synchrotron Radiation Facility (ESRF) in Grenoble at the BioSAXS beamline BM29 using the automated sample changer in flow mode at 20°C, with 10 frames measured, of 1 second duration, per injected sample and an injected volume of 50 μl. The quartz capillary sample cell diameter was 1.8 mm, detector distance 2.867 m, with wavelength λ = 1.008 Å. A buffer baseline was measured before and after each measurement using 50 μl buffer. Before each measurement the protein was diluted into concentrations ranging from 0.2 to 1.8 mg/ml. The size of the particles that is analyzed is limited by the resolution of the instrument. As is the case for the beamline intended for biological SAXS at ESRF, the largest object that can be resolved is 50 nm in size (D_max); thus all particles larger than that will be presented with a size similar to D_max of 50 nm. The average size of the particle is presented as radius of gyration (R_g). Molecular weight of the protein in solution is calculated based the known concentration of protein and the volume calculated. A detailed description of the calculation method can be found at: http://www.esrf.eu/home/UsersAndScience/Experiments/MX/About_our_beamlines/bm29/computing-environment.html.

An outline of the experimental design is presented in Figure 1, indicating the various recombinant AMBN proteins and peptides and test methods used in this study.

The procedure for cloning, expression and purification has previously been described by Wald et al. (2013). The various constructs were wild type, full-length AMBN (AMBN-WT), a variant deleted for exon 5 (DelEx5), a C terminal fragment representing amino acids 223–447 (C-terminus), a N-terminal fragment representing amino acid 27–222 (N-terminus). In addition, peptide representing exon 2–4 (Ex2–4 34 AA, VPFFPQQSGTPGMASLSLETMRQLGSLQRLNTLS), 37 amino acids of exon 5 (Ex5 37 AA, YSRYGFGKSFNSLWMHGLLPPHSSLPWMRPREHETQQ) and Ex5 including the splice variant of exon 6 designated Ex5|Q9NP70 (52 AA, YSRYGFGKSFNSLWMHGLLPPHSSLPWMRPREHETQQYEYSLPVHPPPLPSQ) were included for in vitro phosphorylation in addition to AMBN-WT, DelEx5, N-terminus and C-terminus. All the peptides were in a buffer containing 20 mM Tris-HCl, and 50 mM NaCl. The concentration of the various AMBN proteins and peptides were quantitated using the standard colorimetric Bradford protein assay (Bradford, 1976).

For the DLS experiments the peptide Ex5 37 AA, Ex5|Q9NP70 52 AA and full-length AMBN (AMBN-WT) were employed. Finally, the SAXS experiments were conducted on AMBN-WT, DelEx5, C-terminus. The latter experiments were performed in the presence and absence of Ca²⁺ (0.01 mM).

CK2 phosphorylated AMBN-WT and the C-terminus fragment effectively because dominant bands were observed by SDS-PAGE and phosphoprotein staining. However, neither the AMBN molecule without exon 5 (DelEx5) nor the N-terminus peptide could be effectively phosphorylated with CK2 (Figure 3). PKA phosphorylated both the N-terminus and the C-terminus fragments effectively. On the other hand, PKA was less effective in phosphorylating AMBN-WT and the DelEx5 fragment as visible by the presence of additional significant bands with different molecular weights (Figure 3).

LC–ESI-MS analysis of aliquots from the experiments conducted in Figure 3, revealed a higher incidence of phosphorylation in the C-terminus region of AMBN-WT than in the self-assembled N-terminus and the N-terminus region in AMBN-WT. Moreover, the C-terminus was phosphorylated in additional sites as a region in the self-assembled AMBN-WT than in the C-terminus monomeric peptide (Figure 4 and Table 1).

DLS analyses of peptides Ex5 and Ex5|Q9NP70 indicated no sign of self-assembly (results not shown) confirming the findings reported by Wald et al. (2013). Kinase assays with CK2 and PKA performed on peptides Ex2–4, Ex5, and Ex5|Q9NP70 were analyzed in LC-ESI-MS indicating efficient phosphorylation by PKA. The exons 2–5 encode most of the N-terminal part of AMBN. Yet, the N-terminus fragment was only partly phosphorylated by PKA; both as a self-assembled fragment, and as a region in the full length AMBN-WT. In overall PKA phosphorylated sites in both the N-terminus and the C-terminus part of both the DelEx5 and the full-length AMBN proteins but with un-equal efficacy. This is in accordance with several molecular weights of phosphorylated proteins separated as multiple bands shown in Figure 3 (lanes 3 and 11, respectively). For a detailed overview of phosphorylation sites and incidence in all peptides see Figure 4 and Table 1.

Only AMBN-WT displayed a peak that is typical for folded macromolecules. This feature of AMBN-WT was altered in the presence of Ca²⁺ in all concentrations (Figure 5). DelEx5 had a disordered profile comparable to the C-terminus. The disorder in DelEx5 and C-terminus seemed un-affected by exposure to Ca²⁺ (Figure 5).

SAXS analyzes of AMBN-WT indicated structures above 50 nm in size (D_max) while radius of gyration (R_g) was calculated to 14.5 nm that was that increased upon exposure to Ca²⁺ in all the measured concentrations of AMBN (Table 2). For the self-assembled AMBN with a size larger than the D_max resolution, Rg may here be viewed as the dimensions of AMBN in terms of the width of its fibrils. For DelEx5 and C-terminus within the D_max range, both Rg and D_max may be viewed as dimension of the individual proteins. For DelEx5 and the C-terminus, the Rg was smaller and less affected by Ca²⁺ compared to AMBN-WT. The default settings were employed in calculating Rg for comparison of disordered proteins with fibrillary AMBN proteins (AMBN-WT).

The molecular weight calculated was higher than expected for DelEx5 ranging between 70 and 90 kDa while the C-terminus had close to expected MW ranging between 27 and 43 kDa (Table 2). AMBN-WT was calculated a very high molecular weight, indicative of some sort of supramolecular assembly but these data are restricted by the resolution limit of the instrument at 50 nm (D_max).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MG and handling Editor declared their shared affiliation, and the handling Editor states that the process met the standards of a fair and objective review.