Two-dimensional sum-frequency generation reveals structure and dynamics of a surface-bound peptide

Abstract

Surface-bound polypeptides and proteins are increasingly used to functionalize inorganic interfaces such as electrodes, but their structural characterization is exceedingly difficult with standard technologies. In this paper, we report the first two-dimensional sum-frequency generation (2D SFG) spectra of a peptide monolayer, which are collected by adding a mid-IR pulse shaper to a standard femtosecond SFG spectrometer. On a gold surface, standard FTIR spectroscopy is inconclusive about the peptide structure because of solvation-induced frequency shifts, but the 2D line shapes, anharmonic shifts, and lifetimes obtained from 2D SFG reveal that the peptide is largely α-helical and upright. Random coil residues are also observed, which do not themselves appear in SFG spectra due to their isotropic structural distribution, but which still absorb infrared light and so can be detected by cross-peaks in 2D SFG spectra. We discuss these results in the context of peptide design. Because of the similar way in which the spectra are collected, these 2D SFG spectra can be directly compared to 2D IR spectra, thereby enabling structural interpretations of surface-bound peptides and biomolecules based on the well-studied structure/2D IR spectra relationships established from soluble proteins.

Figure 1

Schematics of possible surface structures of AHP, which is helical in solution. The helical peptide may (a) lie flat on the surface to bury its hydrophobic stripe, (b) denature to maximize sidechain contacts, or (c) stand upright. The G, Y, and sometimes C residues are omitted to highlight the non-thiol sidechains expected to interact most strongly with the gold surface.

Figure 2

Two-dimensional infrared (a–c) and 2D SFG (d–f) spectra of peptide AHP, at waiting times of (a,d) t₂=0, (b,e) 500, and (c,f) 1000 fs. Contours are unfilled between ±20% to emphasize the peaks under discussion. The frequency range of the probe dimension in the 2D IR and 2D SFG spectra are determined by the width of the mid-IR array detector and CCD camera, respectively. The white bar in panels (a) and (d) indicate the nodal line at t₂ = 0.

Figure 3

Comparison of various cuts through the 2D IR and 2D SFG spectra. In (a–c), we give a comparison between diagonal cuts through the fundamental peaks in the 2D IR and 2D SFG spectra (a) t₂=0, (b) 500, and (c) 1000 fs. In (d) and (e), we show the evolution of these cuts as a function of waiting time. In (f), we comapare a horizontal cut through the 2D SFG spectrum at ω_pump = 1660 cm⁻¹ with a cut through the 2D IR spectrum at ω_pump = 1642 cm⁻¹, both with and without broadening by a 25 cm⁻¹ Lorentzian (labeled b25). In all spectra comparing cuts from the 2D IR spectra with cuts from the 2D SFG spectra, the 2D IR cuts have been shifted along the probe axis so that the fundamental lines up with that from the 2D SFG spectrum to enable direct linewidth and lineshape comparisons.

Figure 4

Overlaid pump-probe traces of AHP on gold and in D₂O with fitted exponential decays.

Figure 5

Simulated HD 2D SFG spectra of (a) a 7-residue helix followed by a 5-residue random coil, (b) a 20-residue helix perpendicular to the surface, and (c) a 20-residue helix lying flat on the surface.