Characterizing Protein Structure with Vibrational Probes and Two-Dimensional Infrared Spectroscopy

Abstract

The deposition of β-sheet rich amyloid fibrils is associated with multiple human diseases. Despite being a hallmark characteristic of amyloid-related diseases, there is considerable effort devoted to studying the structures adopted during misfolding that lead to cytotoxic oligomers. Unlike amyloid fibrils, which are incredibly stable, the transient nature of oligomers complicates the characterization of their structures. Probing the amide I’ mode of the peptide backbone with infrared (IR) spectroscopy identifies key secondary structures in peptides and proteins through the assignment of characteristic IR frequency ranges. However, overlapping frequencies in the congested amide I’ region complicate spectral interpretation from only frequency assignments. To overcome spectral congestion, site-specific vibrational reporters, whether in the peptide backbone or through side chain functional groups, provide finer structural detail than bulk amide signals. Two-dimensional infrared (2D IR) spectroscopy, a nonlinear vibrational technique, offers spectral and temporal advantages over linear IR, thereby improving these structural assignments. Thus, in conjunction with site-specific vibrational probes or label-free, 2D IR is a powerful method for examining complex protein structures. This work evaluates labeling schemes for small model peptides and fragments of α-synuclein with azido- or cyano-unnatural amino acids (UAAs) to determine an effective implementation for reporting protein structures in a spectrally transparent region. While labeling schemes provide site specificity, the advancement of transition dipole strength (TDS) analysis, as an extension of 2D IR spectroscopy, has emerged as a robust label-free method for distinguishing protein structures from overlapping spectral features. Through the systematic reporting of TDS values of model peptides accomplished here, TDS analysis was advanced as a tool for quantifying protein secondary structures. This development enabled the direct quantification of the maximum α-helical length and aided in the identification of higher-order structural interactions that otherwise could be obscured in bulk amide 2D IR signals.