Conformation-Selective Ultraviolet–Ultraviolet Hole Burning Spectra of Ubiquitin Ions in a Cryogenic Ion Trap

Abstract

Understanding the structural variations of conformational isomers in proteins is crucial for elucidating protein folding mechanisms. Here, we present a novel method for obtaining conformation-selective ultraviolet (UV)−UV hole burning (HB) spectra of ubiquitin ions ((Ubi+zH) +z , z = 7−10) produced via electrospray ionization. Our approach involves binding multiple N 2 molecules to ubiquitin ions ((Ubi +zH) +z (N 2 ) m , m = 1−55) within a cryogenic ion trap. Upon exposure to UV irradiation, efficient fragmentation of (Ubi +zH) +z (N 2 ) m occurs, primarily yielding bare (Ubi+zH) +z ions as fragments. The significant mass difference between the parent and fragment ions facilitates the acquisition of UV−UV HB spectra, which reveal the presence of at least two distinct conformers. Molecular dynamics simulations suggest that these conformers correspond to A-state structures, differing only in the interactions of a tyrosine residue with neighboring residues. Our findings underscore UV−UV HB spectroscopy of protein ions as a powerful tool for exploring diverse protein isomers.