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 N2 molecules to ubiquitin ions ((Ubi+zH)+z(N2)m, m = 1-55) within a cryogenic ion trap. Upon exposure to UV irradiation, efficient fragmentation of (Ubi+zH)+z(N2)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.
ABSTRACT
Ultraviolet photodissociation (UVPD) spectra of protonated 9-methyladenine (H+9MA), protonated 7-methyl adenine (H+7MA), protonated 3-methyladenine (H+3MA), and sodiated 7-methyladenine (Na+7MA) near the origin bands of the S0-S1 transition were obtained using cryogenic ion spectroscopy. The UV-UV hole burning, infrared (IR) ion-dip, and IR-UV double resonance spectra showed that all the ions were present as single isomers in a cryogenic ion trap. The UVPD spectrum of H+9MA exhibited only a broad absorption band, whereas the spectra of H+7MA, H+3MA, and Na+7MA displayed moderately or well-resolved vibronic bands. Potential energy profiles were computed to understand the reason for the different bandwidths of the vibronic bands in the spectra. The broadening of the bands was correlated with the slopes between the Franck-Condon point and the conical intersection between the S1 and S0 states in the potential energy profiles, thus reflecting the deactivation rates in the S1 state.
ABSTRACT
We obtained ultraviolet photodissociation (UVPD) circular dichroism (CD) spectra of protonated L-phenylalanyl-L-alanine (L-H+PheAla) near the origin band of the S0-S1 transition using cryogenic ion spectroscopy. Infrared (IR) ion-dip, IR-UV hole burning (HB) and UV-UV HB spectra showed that L-H+PheAla existed as two different conformers in a cryogenic ion trap, and they had nearly identical peptide backbones but different conformations in the Phe side chain. The UVPD CD spectra revealed that the two conformers had opposite CD signs and significantly different CD magnitudes from each other. These results demonstrate that the CD value of L-H+PheAla near the origin band is strongly influenced by the conformation of the Phe side chain.
Subject(s)
Dipeptides/chemistry , Circular Dichroism , Density Functional Theory , Ions/chemistry , Photochemical Processes , Protons , Spectrophotometry, Infrared , Ultraviolet RaysABSTRACT
Circular dichroism (CD) spectra contain information about absolute configurations and conformations of chiral compounds. However, extracting this information from CD spectra in solution is challenging, because the spectra exhibit only the averaged CD values of all different conformers. CD spectroscopy of jet-cooled molecules can provide conformation-specific CD spectra, but its application to biomolecules has been limited due to the difficulty of their production in the gas phase. Here, we obtained the first CD spectra of chiral molecular ions produced by electrospray ionization (ESI) using cold ion CD spectroscopy. Protonated l- or d-phenylalanine ions produced by ESI were stored in a cold quadrupole ion trap and irradiated by multiple laser pulses with left or right circular polarization. The CD spectra exhibited well-resolved CD bands of two conformers, whose signs were opposite to each other. This study will broaden the scope of conformation-resolved CD spectroscopy to large molecular ions without size limitations.
