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Secondary and tertiary structures of gaseous protein ions characterized by electron capture dissociation mass spectrometry and photofragment spectroscopy - PubMed

️Tue Jan 01 2002

Secondary and tertiary structures of gaseous protein ions characterized by electron capture dissociation mass spectrometry and photofragment spectroscopy

HanBin Oh et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Over the last decade a variety of MS measurements, such as HD exchange, collision cross sections, and electron capture dissociation (ECD), have been used to characterize protein folding in the gas phase, in the absence of solvent. To the extensive data already available on ubiquitin, here photofragmentation of its ECD-reduced (M + nH)(n-1)+* ions shows that only the 6+ to 9+, not the 10+ to 13+ ions, have tertiary noncovalent bonding; this is indicated as hydrogen bonding by the 3,050-3,775 cm(-1) photofragment spectrum. ECD spectra and HD exchange of the 13+ ions are consistent with an all alpha-helical secondary structure, with the 11+ and 10+ ions sufficiently destabilized to denature small bend regions near the helix termini. In the 8+ and 9+ ions these terminal helical regions are folded over to be antiparallel and noncovalently bonded to part of the central helix, whereas this overlap is extended in the 7+, 6+, and, presumably, 5+ ions to form a highly stable three-helix bundle. Thermal denaturing of the 7+ to 9+ conformers both peels and slides back the outer helices from the central one, but for the 6+ conformer, this instead extends the protein ends away to shrink the three-helix bundle. Thus removal of H2O from a native protein negates hydrophobic interactions, preferentially stabilizes the alpha-helical secondary structure with direct solvation of additional protons, and increases tertiary interhelix dipole-dipole and hydrogen bonding.

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Figures

**Fig 1.**
Number of nonproton deuterium atoms exchangeable in 5+ to 13+ ubiquitin ions: ▴, ref. ; ▾, this study; □ and 5+, 6+ ions (image width indicates abundance), ref. .

**Fig 2.**
Proposed conformational structures for gaseous ubiquitin ions. Black bars, α-helices; shaded areas, H⁺ bound basic residues; dashed loops, possible salt bridges.

**Fig 3.**
ECD spectra of ubiquitin ions at 25°C. Vertical bars: black segment, c ions; open, z⋅ ions; gray filled and open, c and z⋅, respectively, from IRMPD of (M + nH)^(n-1)+• ions. Vertical lines: basic residues of highest protonation probability; estimated intrinsic gas phase basicities of an amino acid in a protein: Arg, 251; His, 237; Lys, 237; and Pro, 227 kcal/mol (38). Some of these data are reprinted with permission from figure 3 of ref. [Copyright (2002) American Chemical Society].

**Fig 4.**
ECD spectra of 6+ to 9+ ions at 155°C. Dashed and dotted vertical lines: higher and lower protonation probability, respectively.

**Fig 5.**
Dissociation of (M + 7H)^6+• and (M + 12H)^11+• ions versus laser irradiation times using 3.0- and 10.6-μm photons, with the 3.0-μm data corrected to be energy equivalent.

**Fig 6.**
Photofragment spectra of (M + 7H)^6+•, (M + 8H)^7+•, and reference ions.

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Secondary and tertiary structures of gaseous protein ions characterized by electron capture dissociation mass spectrometry and photofragment spectroscopy - PubMed