Refining Structural Analysis of Proteins: Automated Methods to Measure Transition Dipole Strength of Single Residues

Transition dipole strength (TDS) analysis enhances two-dimensional infrared (2D IR) spectroscopy by probing protein structural differences that frequency alone cannot resolve. However, its application has been limited to strong signals due to challenges with low signal-to-noise and large backgrounds in the linear optical density. Manual baseline correction can suffer from user error and produce large artifacts that obscure signals of interest. Here, we introduce a new approach incorporating automated baseline correction via the airPLS algorithm to improve the accuracy and precision of TDS calculations across broad spectral windows. Using human islet amyloid polypeptide, we demonstrate TDS analysis of a single ¹³C¹⁸O-labeled residue, enabling a more precise measure of protein structure at the single-residue level. Further, airPLS-corrected TDS spectra can be calculated throughout amyloid aggregation to resolve potential intermediate structures. This work establishes TDS as a robust tool for investigating the structural dynamics of proteins and other complex macromolecular assemblies.