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

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 = 7-10) produced via electrospray ionization. Our approach involves binding multiple N molecules to ubiquitin ions ((Ubi+zH)(N), m = 1-55) within a cryogenic ion trap. Upon exposure to UV irradiation, efficient fragmentation of (Ubi+zH)(N) occurs, primarily yielding bare (Ubi+zH) 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.