Photocontrolled Reversible Amyloid Fibril Formation of Parathyroid Hormone-Derived Peptides

Peptide fibrillization is crucial in biological processes such as amyloid-related diseases and hormone storage, involving complex transitions between folded, unfolded, and aggregated states. We here employ light to induce reversible transitions between aggregated and nonaggregated states of a peptide, linked to the parathyroid hormone (PTH). The artificial light-switch 3-{[(4-aminomethyl)phenyl]diazenyl}benzoic acid (AMPB) is embedded into a segment of PTH, the peptide PTH25–37, to control aggregation, revealing position-dependent effects. Through in silico design, synthesis, and experimental validation of 11 novel PTH25–37-derived peptides, we predict and confirm the amyloid-forming capabilities of the AMPB-containing peptides. Quantum-chemical studies shed light on the photoswitching mechanism. Solid-state NMR studies suggest that β-strands are aligned parallel in fibrils of PTH25–37, while in one of the AMPB-containing peptides, β-strands are antiparallel. Simulations further highlight the significance of π–π interactions in the latter. This multifaceted approach enabled the identification of a peptide that can undergo repeated phototriggered transitions between fibrillated and defibrillated states, as demonstrated by different spectroscopic techniques. With this strategy, we unlock the potential to manipulate PTH to reversibly switch between active and inactive aggregated states, representing the first observation of a photostimulus-responsive hormone.


Supplementary figures
Figure S1.The smoothed potential energy curves from 34 converged cis ↔ trans isomerization paths.Single-point calculations are performed using NEVPT2(2,2)/xTB with implicit water through ALPB.The smoothing is carried out using Gaussian process regression.A subset of the paths has an energy barrier in the trans-cis-isomerization, notably paths 04, 10 and 21, with excitation energies before the barrier range from 2.5 to 3.0 eV.This correlates with the experimental fluorescence peak at 485 nm (Figure S2), albeit with an expected ipsochromic effect caused by the minimum active space considered in the calculations.We attribute the extended fluorescence lifetime, compared to azobenzene, to structures hindered by this barrier, preventing radiationless relaxation.These barriers predominantly arise from structures characterized by dihedral angles around 170°, which exhibit a heightened S0-S1 excitation energy in the scan.Figure S3.All geometries with dihedral angles near 170° projected on to the three-dimensional PCA space.The colors encode the S0-S1 excitation energies.The reduced three-dimensional space accounts for 86% of the total variance.Several clusters can be detected, notably one predominantly comprising structures with high excitation energies (visually represented in dark red in the lower left corner of the projection).This particular cluster is positioned at the negative extremity of the PC1-axis, advances towards the positive end of the PC2-axis and is situated approximately mid-way along the PC3-axis.   C spin diffusion spectra of fibrils from PTH25−37, uniformly 13 C labeled for L28 and F34, recorded under different conditions.Red: magnetic field strength 14.1 T (corresponding to 600 MHz 1 H resonance frequency), spinning speed 11 kHz, mixing time 10 ms.Blue: magnetic fiels strength of 18.8 T (corresponding to 800 MHz 1 H resonance frequency), spinning frequency of 20 kHz, mixing time 1 s. Figure S6 shows the 2D 13 C, 13 C spin diffusion spectra of fibrils from PTH25−37, uniformly 13 C labeled for L28 and F34, recorded under different conditions.In the red spectrum, intraresidual cross-peaks between neighboring 13 C sites of L28 and F34, respectively, are visible.At a longer mixing time of 1 s (blue), long-range correlations between all 13 C spins within one residue are obtained.Spin systems of the labeled amino acids F34 (cyan) and L28 (brown) are marked by solid lines.Dashed lines show a possible contact between L28 Cγ/Cδ and an aromatic F34 carbon.The spinning speed of 20 kHz corresponds to the first order rotational resonance condition for resonances with a chemical shift difference of 100 ppm, leading to a recoupling of dipolar couplings between aromatic ring carbon atoms of F34 and aliphatic Cγ and Cδ signals of L28, facilitating magnetization transfer between those residues if the distance between these residues would not exceed 6 Å. 20 The fact that no inter-residual cross-peaks between L28 and F34 can be observed is thus a strong indication against an antiparallel arrangement of β-strands within the β-sheet.

Figure S2 .
Figure S2.Fluorescence life-time measurements.Fluorescence spectra measured at various points in time after the excitation pulse.

Figure S4 .
Figure S4.(a) The first three principal components represented in the distance matrix space.The first principal component signals a considerable separation between the residues R25 and A36/L37.Given this cluster's positioning at the PC1-axis's negative end, it implies that within these geometries, the distances between these residues are notably reduced.The second principal component reveals negative values for the separation between R25 and H32/N33, alongside positive distances between D30 and L37.The positive projection of this cluster on the PC2 suggests these distance matrix characteristics directly, without reversing the sign.With almost negligible projection on PC3, this component scarcely influences the distance matrices for structures within this cluster.(b) Distance matrices for clusters with high excitation energies.Despite originating from different paths, these matrices exhibit consistent patterns.

Figure S5 .
Figure S5.Selected monomer structure (left) with distance matrix shown in FigureS4band dimer structure (right) with a similar distance matrix.The corresponding distance matrices are shown in the upper right corners.It can be seen that the simultaneous interactions of R25 with A36/L37 and H32/N33 potentially contribute to the S1-barrier during the trans→cis isomerization path, leading to longer fluorescence lifetimes.This structural motif is also found in the dimer, while their configurations stabilized by intermolecular interactions could also lead to an increased barrier.

Figure S7 .
Figure S7.2D 13 C,13  C spin diffusion spectra of fibrils from PTH25−37, uniformly13  C labeled for L28 and F34, recorded under different conditions.Red: magnetic field strength 14.1 T (corresponding to 600 MHz 1 H resonance frequency), spinning speed 11 kHz, mixing time 10 ms.Blue: magnetic fiels strength of 18.8 T (corresponding to 800 MHz 1 H resonance frequency), spinning frequency of 20 kHz, mixing time 1 s.FigureS6shows the 2D 13 C, 13 C spin diffusion spectra of fibrils from PTH25−37, uniformly 13 C labeled for L28 and F34, recorded under different conditions.In the red spectrum, intraresidual cross-peaks between neighboring 13 C sites of L28 and F34, respectively, are visible.At a longer mixing time of 1 s (blue), long-range correlations between all 13 C spins within one residue are obtained.Spin systems of the labeled amino acids F34 (cyan) and L28 (brown) are marked by solid lines.Dashed lines show a possible contact between L28 Cγ/Cδ and an aromatic F34 carbon.The spinning speed of 20 kHz corresponds to the first order rotational resonance condition for resonances with a chemical shift difference of 100 ppm, leading to a recoupling of dipolar couplings between aromatic ring carbon atoms of F34 and aliphatic Cγ and Cδ signals of L28, facilitating magnetization transfer between those residues if the distance between these residues would not exceed 6 Å.20  The fact that no inter-residual cross-peaks between L28 and F34 can be observed is thus a strong indication against an antiparallel arrangement of β-strands within the β-sheet.

Figure S8 .
Figure S8.2D 13 C, 13 C spin diffusion spectra of fibrils from P4, uniformly 13 C labeled for L28 and F34, recorded at a magnetic field strength of 18.8 T (corresponding to 800 MHz 1 H resonance frequency) at a spinning speed of 20 kHz, corresponding to the first order rotational resonance condition for signals with a chemical shift difference of 100 ppm.Red: Mixing time of 50 ms.Blue: Mixing time of 1 s.See for more information in Fig. S6.Here, inter-residual cross-peaks between the aromatic ring signals of F and L C resonances are clearly visible for a mixing time of 1 s (blue spectrum).

Figure S9 .
Figure S9.Summary of all MD simulation results.The average numbers of residues ⟨Nres⟩ forming secondary structure elements, divided into α-helical (magenta), intrapeptide β-sheets (blue), interpeptide parallel β-sheets (cyan) and interpeptide antiparallel β-sheets (green) are given.The average was taken over the number of frames of the simulation and normalized by the number of peptides NP present in the corresponding simulation.

Figure S10 .
Figure S10.Electrostatic potential surface of PTH25−37 and the peptides P1, P3, P4, P8, and P12, with values according to the color map at the bottom, ranging from -2 (red) to +2 kTe −1 (blue).The electrostatic potential mapped to the molecular surfaces was calculated using the Adaptive Poisson-Boltzmann Solver (APBS 1 ) plugin for the pymol 2 software package.For each peptide, the two views that are rotated by 180° around the backbone axis are shown, as well as a transparent front view.

Figure S11 :
Figure S11: Control experiment to investigate photobleaching of the azobenzene unit during the photoisomerization.Absorption of P4 was measured after each isomerization step at 327 nm (top, blue dots) and 295 nm (bottom, red dots).Light red area corresponds to irradiation with light of 340 nm wavelength for 30 min to achieve trans→cis isomerization.Light blue area corresponds to irradiation with light of 405 nm wavelength for 30 min to achieve cis→trans isomerization.

Figure S21 .
Figure S21.TEM images of P1 at different times and temperatures; scale bar = 250 nm.

Figure S22 .
Figure S22.TEM images of P2 at different times and temperatures; scale bar = 250 nm.

Figure S23 .
Figure S23.TEM images of P4 at different times and temperatures; scale bar = 250 nm.

Figure S24 .
Figure S24.TEM images of P7 at different times and temperatures; scale bar = 250 nm.

Figure S25 .
Figure S25.TEM images of P8 at different times and temperatures; scale bar = 250 nm.

Figure S26 .
Figure S26.TEM images of P9 at different times and temperatures; scale bar = 250 nm.

Figure S27 .
Figure S27.TEM images of P12 at different times and temperatures; scale bar = 250 nm.

Figure S28 :
Figure S28: CD-spectra of the reversible fibrillization of P4 over three cycles (1 st , 2 nd , and 3 rd ).trans-Isomer (blue) was measured directly after dissolving the peptide, trans-fibrils (green) were measured 20 h after reference sample reached the stationary phase, and irradiated fibrils (orange) were measured directly after irradiating the fibrils with 340 nm for 5 h.

Figure S29 :
Figure S29: UV/Vis-spectra of trans-P4 monomer before each fibrillization cycle.Dashed line corresponds to the absorption maxima of the trans-isomer at 327 nm.

Table S1 .
All Simulations performed, with their respective simulation time

Table S2 .
Sequence of PTH25-37 and P1 -P12 and their respective solubility in buffered solution (50 mM aqueous Na2HPO4) with pH 7.4, the critical fibrillization concentration (ccr), and standard free energy of the fibrillization reaction ΔG 0 .