Probing the unfolding of myoglobin and domain C of PARP-1 with covalent labeling and top-down ultraviolet photodissociation mass spectrometry.
Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top-down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed electron transfer dissociation (ETD) in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation in the present study support that PARP-C exists as a monomer in solution with a close-packed C-terminal α helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins.
Department of Chemistry, University of Texas at Austin , 1 University Station A5300, Austin, Texas 78212, United States.
Michael
M
Cammarata
Ke-Yi
KY
Lin
Jeff
J
Pruet
Hung-Wen
HW
Liu
Jennifer
J
Brodbelt
D000818:Animals; D006736:Horses; D013058:Mass Spectrometry / Q000379:methods*; D009211:Myoglobin / Q000737:chemistry*; D055668:Photochemical Processes; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry*; D058767:Protein Unfolding; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D009211:Myoglobin; D011065:Poly(ADP-ribose) Polymerases