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. 2022 Jan 30;434(2):167385.
doi: 10.1016/j.jmb.2021.167385. Epub 2021 Dec 6.

Conformational Tuning of Amylin by Charged Styrene-Maleic-Acid Copolymers

Bikash R Sahoo  1 , Christopher L Souders 2nd  2 , Takahiro Watanabe-Nakayama  3 , Zhou Deng  4 , Hunter Linton  5 , Saba Suladze  6 , Magdalena I Ivanova  7 , Bernd Reif  6 , Toshio Ando  3 , Christopher J Martyniuk  8 , Ayyalusamy Ramamoorthy  9
Affiliations

Conformational Tuning of Amylin by Charged Styrene-Maleic-Acid Copolymers

Bikash R Sahoo et al. J Mol Biol. .

Abstract

Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene-maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are α-helical, the ones formed with SMAEA are rich in β-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed π-π interaction with Phe23, electrostatic π-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylin's C terminus (residues 31-37) that belongs to one of the two β-sheet regions (residues 14-19 and 31-36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species.

Keywords: IAPP; SMA copolymer; amylin; amyloid; type-II diabetes.

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Conflict of interest statement

Declaration of interests The polymers used in this study SMAEA and SMAQA are produced in the Ramamoorthy lab at Michigan. They are US patented.

Figures

Figure 1.
Figure 1.
HS-AFM images showing the growth of human-amylin fibrils on a mica stage using freshly dissolved 5 μM amylin monomers which was mixed with preformed fibrils deposited on the mica surface. Arrow indicates fast-growing (arrow head) and slow-growing (arrow tail) ends of a selected amylin fiber seed. The de novo formed fibers are circled.
Figure 2.
Figure 2.
The effect of SMA copolymers on amylin fibrillation monitored by real-time HS-AFM. HS-AFM images of 5 μM amylin dissolved in 30 mM NaAc, pH 5.5 in the presence of 11 μg/mL SMAQA (a) and SMAEA (b). The de novo globulomer formation in the presence of SMAQA is shown inside a white box (in (c) and (e)). The size of the globulomers (as shown in (d) and (f)) were determined using ImageJ where on the x-axis 1pixel=2 nm. The bidirectional fibril growth in the presence of SMAEA is shown by arrows at t=225 s (g), and the time lapse images of fast and slow growing ends. (h) The de novo globulomer formation is shown inside a white box in (g). The growth of de novo globulomer into fibers in the presence of SMAEA is shown inside the white box in (i).
Figure 3.
Figure 3.
(a) Monitoring the change in thioflavin-T (10 μM) fluorescence intensity in the presence of SMA copolymers as a function of amylin fiber concentration (see methods). Fluorescence intensity of samples containing no amylin fiber is measured in the initial 10 minutes. Preformed amylin fibers (1%, 2% and 7% (v/v)) (except the black curve for ThT alone) were added at time-intervals 10, 20 and 40 minutes, respectively. (b-c) Thioflavin-T fluorescence spectra of 10 μM human-amylin (4 replicates) dissolved in NaAc buffer, pH 5.5 in the absence (black) or presence (red: 5.5 μg/mL; blue: 11 μg/mL; green: 22 μg/mL; purple: 44 μg/mL) SMAQA (b) or SMAEA (c). (d) 2D plot showing the fluorescence of 10 μM of ThT (only), and ThT mixed with 5 μM amylin monomers (labeled as mAmylin), preformed fibers (fAmylin), filtered SMAQA (11 μg/mL)+ 5 μM amylin (labeled as globulomer), and filtered SMAEA (11 μg/mL)+ 5 μM amylin (labeled as Fiber) (see Figure S1).
Figure 4.
Figure 4.
(a) Time-lapse far-UV CD measurement of 25 μM amylin dissolved in NaAc buffer, pH 5.5 in the presence and absence of 55 μg/mL SMAEA or SMAQA (~15 min:solid circle; ~24 h:open circle) (b) FT-IR spectra show the effect of copolymers on the secondary structure of human-amylin. 25 μM of freshly dissolved human-amylin mixed with 55 μg/mL SMAQA or SMAEA was incubated for ~12-hours following lyophilization and used for FT-IR measurement. Region spanning 1600–1700 cm−1 representing the peptide secondary structure is highlighted and zoomed (box). Freshly dissolved amylin monomers and fibers prepared from 25 μM monomers following ~72 hours continuous agitation at room temperature were lyophilized and used as a control for comparative conformational analysis. (c) 2D 15N/1H SOFAST-HMQC spectra of 25 μM 15N-labeled amylin in the absence (red) and presence of 55 μg/mL SMAQA (green) or SMAEA (blue) recorded on an 800 MHz NMR spectrometer at 25 °C. (d) Peak intensities measured from SOFAST-HMQC spectra: peak intensities of amylin residues in the absence (I0) and in the presence of polymer (I): SMAEA (blue) and SMAQA (green). * indicates peak intensities from I26/V32 in the SMAEA-amylin sample. (e) Chemical shift perturbations (CSPs) calculated using ΔδNH=δ1H2+0.154×δ15N2. The dashed red line represents the average (CSPavg) value calculated for the amylin-SMAQA mixture, whereas CSPavg is not shown for amylin-SMAEA due to significant loss of signal intensity for most of the residues.
Figure 5.
Figure 5.
Structural interactions between amylin and SMA copolymers. (a-b) Time-lapse 1H NMR spectra of 55 μg/mL polymer (black) mixed with 25 μM amylin (blue and red) recorded on a 500 MHz NMR spectrometer at 25 °C at the indicated times. The styrene 1H peak is highlighted in yellow in (b). A broad low intensity peak from amylin oligomers appeared near ≈−0.5 ppm is indicated with *. The SMAEA-amylin solution showed precipitation whereas the SMAQA-amylin samples were clear at the end of day-7 as shown in the NMR tubes (top, center). (c-d) STD 1H NMR spectra of 25 μM amylin in the presence of 55 μg/mL SMAQA (c) and SMAEA (d) recorded on a 500 MHz NMR spectrometer at 25 °C. The reference spectrum is shown in black and the saturation transfer difference spectrum is shown in the indicted colors (blue or pink) and the saturated peaks are indicated by arrows. x-axis offset is used for the cyan spectrum to avoid spectral overlapping and for visual clarity. Snapshots obtained at 500 ns MD simulations are shown for SMAQA-amylin (e) and SMAEA-amylin (f) complexes. The interaction between polymer (ball-stick) with amylin (cartoon) shown in the structures that were generated using DSV v17.2.0. The amylin binding residues that form hydrogen bonding, electrostatic, hydrophobic and π-π interactions are labeled (shown as dashed lines). The top and bottom views of the hydrophobic surface maps of the complexes are shown on the right of the respective complex structure.
Figure 6.
Figure 6.
Modulation of amylin toxicity by SMA copolymers. (a) HEK-293T cell viability using MTT assay. Cells treated with SMA copolymer (EA or QA) incubated with/without 10μM of amylin at the indicated polymer concentrations. All samples were normalized using the MTT absorbance of cells incubated with 30mM NaAc buffer, pH 5.5. All quantitation results are shown as mean ± SEM for 3 technical (n) replicates and 5 independent (N) experiments; statistical analyses were done using Kruskal-Wallis test. The filtered amylin species obtained from the peptide-amylin mixture is denoted as EA (f) or QA (f). (b-d) % survival (b) and % deformity (c) of zebrafish embryo (N=3, n=15) treated with samples (10μM amylin; 22μg/mL polymers) indicated in color as a function of days-post-fertilization (dpf). (d) The statistical results are obtained from the images showing deformity in zebrafish embryo taken at different time-points. Scale= 2000 μm.
Scheme 1.
Scheme 1.
(a) Chemical structures of styrene-maleic acid copolymers SMAQA and SMAEA. (b) Amino acid sequence of human-amylin.
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