Acidic pH Promotes Refolding and Macroscopic Assembly of Amyloid β (16–22) Peptides at the Air–Water Interface

Assembly by amyloid-beta (Aβ) peptides is vital for various neurodegenerative diseases. The process can be accelerated by hydrophobic interfaces such as the cell membrane interface and the air–water interface. Elucidating the assembly mechanism for Aβ peptides at hydrophobic interface requires knowledge of the microscopic structure of interfacial peptides. Here we combine scanning force microscopy, sum-frequency generation spectroscopy, and metadynamics simulations to probe the structure of the central fragment of Aβ peptides at the air–water interface. We find that the structure of interfacial peptides depends on pH: at neutral pH, the peptides adopt a less folded, bending motif by forming intra-hydrogen bonds; at acidic pH, the peptides refold into extended β-strand fibril conformation, which further promotes their macroscopic assembly. The conformational transition of interfacial peptides is driven by the reduced hydrogen bonds, both with water and within peptides, resulting from the protonation of acidic glutamic acid side chains.


Scanning Force Microscopy (SFM):
Sample preparation: Atomically flat mica substrates were placed inside a home built Teflon trough.
Aβ16-22 peptide solution in 5 mL volume at pH 3 and 7 were injected into trough, the peptide solutions were equilibrated for 2 hours, which ensures the fully adsorption and assembly of peptides at the air-water interface. Bulk peptide solution (below interfacial peptides) was removed slowly and carefully using a syringe, which was connected to the pinhole at the bottom of the trough. The bottom plane of the Teflon trough is tilted with respect the horizontal water surface, this geometry allows the removal of bulk peptides, while results in the deposition of interfacial peptides onto mica surface. The deposited peptide layers on mica were subsequently measured by SFM.
SFM measurements were performed with a commercial instrument (Bruker Dimension ICON) operated in tapping mode (OTESPA, with a nominal resonance frequency of 300 kHz and a spring constant of 26 N/m) in air.

Surface Pressure measurements
Surface pressure has been measured using a Langmuir tensiometer (Kibron, Finland). The Teflon trough was thoroughly cleaned sequentially with acetone, ethanol, and milliQ water, and dried under a nitrogen stream prior to measurements. The surface pressure (π) was normalized with pure water to 0 mN/m.

Vibrational Sum Frequency Generation (SFG) spectroscopy
Homodyne SFG: The vibrational SFG spectra were obtained by overlapping, in time and space, the visible and IR pulses. A Ti:Sapphire amplified system (Spitfire Ace, Spectra Physics Inc.) delivers 35 fs long pulses at a central wavelength of ~800 nm and 1 KHz repetition rate. The beam is split in two parts: one it is spectrally narrowed using a Fabry-Perot etalon to achieve spectral resolution of 15 cm -1 (lambda=800 nm, E~25 mJ/pulse). The other part is used to generate tunable broadband IR pulses thanks to a parametric optical amplifier followed by a noncollinear difference frequency generation module (TOPAS Prime). The average power is 2 J/pulse at a wavelength of 6000 nm and 3 J/pulse at a wavelength of 3000 nm. Visible and IR beams are focused onto the sample using respectively a 20 cm and 5 cm focal length (FL) lenses. The polarization of both beams can be controlled (S or P) with a polarizer and a half waveplate. Beams are temporally and spatially overlapped at the sample position. The SFG signal is generated with Visible and IR beam angles of 55° and 60° respective to the surface normal, and the signal is collimated using a 20 cm FL lens, and focused into a spectrograph using a 5 cm FL achromatic lens, dispersed by a grating and collected by an Electron-multiplying CCD (EMCCD) camera. The polarization of the SFG signal can be well controlled like Visible and IR beams.
Each SFG spectrum was acquired for 10 minutes, and the spectra are normalized by non-resonance reference spectra of z-cut quartz crystal after background correction. Spectra were recorded in the SSP (sum, visible, and infrared) or PSP polarization combination. For SFG experiments in amide I region, D2O solvent was used to avoid the spectra interference from the bending mode of H2O, and spectra were calibrated by the absorption bands of water vapor. Spectra recorded in CH/OH region were referenced by the absorption bands of polystyrene.
SFG spectra were fitted by Lorentzian peak shapes according to the following equation: In equation (1) above, the susceptibility (2) consists of a non-resonant ( (2) ) and a resonant ( (2) ) term. ANR and NR are the amplitude and phase of non-resonant signal, respectively. An is the amplitude of resonant signal, n is the resonant frequency, IR is the infrared frequency, and Γn is the width of transition.
Heterodyne SFG: In heterodyne detection, SFG signals are generated from both local oscillator (LO) and from the sample. The two SFG signals are delayed in time with respect to each other by passing the LO SFG beam through a silica plate. The two SFG beams are sent into a monochromator and detected by EMCCD. The interference pattern of the two SFG signals are analyzed using a Fourier transformation. The spectra in time domain was processed using rectangular function, finally both the real and imaginary parts of χ (2) (Imχ (2) ) can be extracted, by referencing the heterodyne SFG signal of the sample with that for zcut quartz whose SFG phase is already known. ) 44 a  ssymmetric stretching,  asasymmetric stretching, bending motion,  sc -scissor Table S2. Peak fitting parameters and assignment for PSP SFG spectra in Figure 2b, for the A 16-22 peptides at water surface with solution pH of 3 and 7.   [ (11]. Aβ16-22 peptides were placed at the water/vacuum interfaces of a water slab of 60x60x100 A 3 (see Figure S); 6 Cland 2 Na + ions were added to pH 3, whereas 2 Cland 2 Na + were added to pH 7. Aβ16-22 was treated with the classical force field OPLS [(9], while the SPC/E model[ (2] was used for water molecules, as it was shown that this model was reliable for describing the water/vacuum interface with sufficient accuracy [ (11].
The simulations were conducted using periodic boundary conditions (PBC) in all the three dimensions; nevertheless, the long-range part of the electrostatic potential were treated with Particle-Mesh-Ewald (PME) [5] taking in account the periodicity only along x and y directions by exploiting the pseudo-2d Ewald summation (3DC)[ (12]. The system had a slab geometry and the dimension of x-y plane was the same of the water box. The z-dimension, accordingly to the reduced Ewald-geometry[ (12], was set three times larger than the height of the water slab. The final simulation box was 60x60x360 Å 3 for both systems. The Fourier spacing for the PME summation was set 1.2 Å whereas the distance cut off for non-bonded interactions was set to 13 Å. All MD simulations were performed in the NVT ensemble (T=300 K) controlled via the stochastic velocity-rescaling thermostat.[(4] Integration time step was set to 2 fs and all bonds were treated as holonomic constraints using the LINCS algorithm [6]. Both systems were minimised and subjected to 2 ns of MD simulations. The final conformations were used to perform 430 ns of well tempered metadynamics [1] exploiting as a collective variable the dihedral angle defined by (CB-CA)-(CA-CB) atoms of the adjacent phenyl-alanine residues ( Figure S). The width of the Gaussian function was 0.2 rad and the initial height of the Gaussian functions was 1.5 kJ/mol. The "biasfactor" for well-tempered metadynamics was set to 15. The bias-potential was regularly updated at every 4 ps intervals throughout the simulations.    hydrophobic side chains agree well with that revealed from the heterodyne SFG spectra in Figure  Figure S6: Distribution of the location (z) of Aβ16-22 peptides at two pH. The location z-distance is defined for the center of mass (COM) of peptides with respect to the interface (see Figure S3 (a)).