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

Area of Science:

• Surface Science
• Spectroscopy
• Biophysics

Background:

• Functionalizing inorganic surfaces with proteins and polypeptides is crucial for applications like biosensors and advanced materials.
• Characterizing the structure of these surface-bound biomolecules is challenging with conventional spectroscopic techniques due to interface complexities and solvation effects.

Purpose of the Study:

• To develop and apply a novel spectroscopic method for detailed structural analysis of peptide monolayers on inorganic surfaces.
• To demonstrate the capability of two-dimensional sum-frequency generation (2D SFG) spectroscopy in elucidating the conformation of surface-immobilized peptides.

Main Methods:

• Implementation of a mid-infrared pulse shaper with a femtosecond SFG spectrometer to acquire 2D SFG spectra.
• Analysis of 2D SFG spectral line shapes, anharmonic shifts, and vibrational lifetimes to infer molecular structure.
• Comparison of 2D SFG spectra with traditional Fourier-transform infrared (FTIR) spectroscopy to highlight advantages.

Main Results:

• 2D SFG spectra revealed that the peptide monolayer on a gold surface adopts a predominantly α-helical and upright conformation.
• The technique successfully detected random coil residues through cross-peaks in the 2D SFG spectra, despite their isotropic distribution.
• Solvation-induced frequency shifts, which obscure structural information in FTIR, were overcome by the 2D SFG approach.

Conclusions:

• 2D SFG spectroscopy is a powerful tool for characterizing the structure of surface-bound peptides and proteins.
• The established structure-2D IR spectra relationships for soluble proteins can be extended to surface-bound systems via 2D SFG.
• This advancement facilitates rational peptide design for functionalized interfaces and biomolecular applications.