Chiral Sum Frequency Generation for In Situ Probing Proton Exchange in Antiparallel β-Sheets at Interfaces

Abstract

Studying hydrogen/deuterium (H/D) exchange in proteins can provide valuable insight on protein structure and dynamics. Several techniques are available for probing H/D exchange in the bulk solution, including NMR, mass spectroscopy, and Fourier transform infrared spectroscopy. However, probing H/D exchange at interfaces is challenging because it requires surface-selective methods. Here, we introduce the combination of in situ chiral sum frequency generation (cSFG) spectroscopy and ab initio simulations of cSFG spectra as a powerful methodology to probe the dynamics of H/D exchange at interfaces. This method is applied to characterize H/D exchange in the antiparallel β-sheet peptide LK7β. We report here for the first time that the rate of D-to-H exchange is about 1 order of magnitude faster than H-to-D exchange in the antiparallel structure at the air/water interface, which is consistent with the existing knowledge that O-H/D dissociation in water is the rate-limiting step, and breaking the O-D bond is slower than breaking the O-H bond. The reported analysis also provides fundamental understanding of several vibrational modes and their couplings in peptide backbones that have been difficult to characterize by conventional methods, including Fermi resonances of various combinations of peptide vibrational modes such as amide I and amide II, C-N stretch, and N-H/N-D bending. These results demonstrate cSFG as a sensitive technique for probing the kinetics of H/D exchange in proteins at interfaces, with high signal-to-noise N-H/N-D stretch bands that are free of background from the water O-H/O-D stretch.

Biological Macromolecules at Interfaces Probed by Chiral Vibrational Sum Frequency Generation Spectroscopy

“…70 For example, Figure 8A shows the chiral SFG spectrum of the peptide of LK 7 β obtained using the psp polarization setting at the air/water interface. 84 The peptide, with a sequence of LKLKLKL, is highly amphiphilic and is known to form antiparallel β-sheet at the air/water interfaces. 102 The peptide was dissolved in phosphate buffer (10 mM, pH 7.4).…”

“…The observation of protein backbone chiral N−H stretch with zero water background has introduced the opportunity of probing real-time H/D exchange in protein at interfaces. 84 The experiments were performed using a model system of the amphiphilic LK 7 β peptide, which forms antiparallel β-sheet structures at the air/water interface ( Figure 16A). This peptide exhibits chiral SFG spectra in amide I regions at the air/H Figure 16B This work demonstrates a novel application of chiral SFG: probing H/D exchange of proteins at interfaces in situ and in real time.…”

Characterization of Parallel β-Sheets at Interfaces by Chiral Sum Frequency Generation Spectroscopy

“…26–28 We have monitored, in situ and in real time, the misfolding of human islet amyloid polypeptide (hIAPP) into parallel β -sheets via an α-helical intermediate at the lipid/water interface, 26, 28–29 and proton exchange in parallel β–sheets at interfaces. 30 We have also demonstrated that chiral SFG can be used to determine the orientation of the parallel β -strand of hIAPP aggregates at interfaces by analyzing the chiral amide I spectrum. 28, 31–32 …”

Communication: Probing the absolute configuration of chiral molecules at aqueous interfaces

“…[8][9][10][11][12][13][14] While CD-spectroscopy is a linear optical technique, the enantiomer-specificity of which results from inherently weak magnetic dipole or electric quadrupole interactions, the potential of non-linear optical techniques such as second harmonic generation (SHG) and sum-frequency generation (SFG) to study chiral molecules has been increasingly recognized over the past years. [15][16][17][18][19][20][21][22][23][24][25] Vibrational SFG-spectroscopy (VSFG) is a second-order non-linear optical technique, in which an infrared pulse and a visible pulse are combined to generate light at their sum-frequency. This generation is enhanced in case the infrared light is in resonance with specific molecular vibrations.…”

“…Recently, chiral VSFG-spectra have been successfully measured for protein monolayers in the spectral region of the amide I vibration (C= =O-stretch) and the NH-stretch vibration, 19,[21][22][23] which allowed, e.g., to obtain unprecedented insights into the aggregation mechanism of amyloidic peptides at water-lipid interfaces. 22 While these studies have focussed on the intensity of the generated SFG-light I s f g ∝ | χ (2) | 2 , the sign of the non-linear susceptibility χ changes between enantiomers, and thus, information on the absolute configuration of chiral molecules can be obtained if the real and imaginary parts of χ (2) chiral are determined.…”