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Studies of noncovalent protein-protein and protein-small-molecule complexes by electrospray ionization mass spectrometry Kang, Yang

Abstract

Gas-phase ions of tetrameric protein−protein complexes, hemoglobins, and protein−small-molecule complexes, cellulasexylanase (Cex) binding with aza-sugar inhibitors, have been studied by electrospray ionization mass spectrometry with measurements of collision cross sections, hydrogen/deuterium exchange (H/Dx), and tandem mass spectrometry (MS/MS), with the goal of investigating their gas-phase conformations and binding strengths and any relation to the solution properties. Adult hemoglobin (Hb A, α₂β₂) prepared from fresh erythrocytes and commercial lyophilizate showed similar collision cross sections, but different mass spectra. Heme-deficient dimers found in commercial Hb are attributed to oxidative modifications on the β chains. Both oxidation and lyophilization in commercial proteins produce higher levels of monomer and dimer ions in mass spectra. Gas-phase properties of ions of Hb A, sickle hemoglobin (Hb S, E6V[β]) and fetal hemoglobin (Hb F, α₂γ₂) have been compared. Tetramer ions of Hb S have greater collision cross sections and higher gas-phase H/Dx levels than Hb A tetramers. In MS/MS, dimer and tetramer ions of Hb F show different dissociation pathways and energies from the same species of Hb A and S. Thus the single mutation in the β chain of Hb S and the 39 residue alterations of the γ chain of Hb F change the gas-phase properties of Hb complexes. No consistent correlation is found between gas-phase stability and solution binding constants. With a home-built “time-resolved” ESI source, millisecond-H/Dx in solution is studied with these three hemoglobins. No conformational differences are found. Interestingly, H/Dx kinetics show good correlations with tetramer-dimer (ca. 1 s) and dimer-monomer (minutes) inter-conversions in solution. In gas-phase ions of three Cex−inhibitor complexes, Cex−xylobiosyl deoxynojirimycin (X₂DNJ), Cex−xylobiosyl isofagomine lactam (X₂IL), and Cex−xylobiosyl isofagomine (X₂IF), the relative binding strengths measured by MS/MS follow the order in solution. In solution complexes have less flexibility and therefore exchange fewer hydrogens than free proteins. In the gas phase, complexes have lower collision cross sections than free proteins, indicating a more compact structure, but interestingly complexes show greater H/Dx levels. Detailed studies suggest that ions can “remember” their original solution conformations for milliseconds in the gas phase, but may fold or unfold on a time scale of seconds.

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