Elucidating the tertiary structure of protein ions in vacuo with site specific photoinitiated radical reactions

Abstract

A new method for identifying residue specific through space contacts as a function of protein secondary and tertiary structure in the gas phase is presented. Photodissociation of a non-native carbon-iodine bond incorporated into Tyr59 of ubiquitin yields a radical site specifically at that residue. The subsequent radical migration is shown to be highly dependent on the structure of the protein. Radical-directed dissociation (RDD) of low charge states, which adopt compact structures, generates backbone fragmentation that is prominently distributed throughout the protein sequence, including residues that are distant in sequence from Tyr59. Higher charge states of ubiquitin, which adopt elongated, unfolded structures, yield RDD that is primarily nearby in sequence to Tyr59. Regardless of which structure is probed, information at the residue-level is obtained by examining specific radical-donor and radical-acceptor pairs. The relative importance of a particular interaction pair for a specific conformation can be revealed by tracking the charge state dependence of the dissociation. Structurally important contact pairs exhibit strong and concerted changes in relative intensities as a function of charge state and can also be used to reveal structural features which persist among different protein structures. Moreover, incorporation of distance constraint information into molecular mechanics conformational searches can be used to drive the search toward relevant conformational space. Implementation of this approach has revealed highly stable, previously undiscovered structures for the +4 and +6 charge states of ubiquitin, which bear little resemblance to the crystal structure.

Figure 1

a) Photodissociation of the +5 charge state of iodoubiquitin cleaves the iodine atom, yielding a protein radical ion. b) Collision induced dissociation of the radical product from (a), yields prominent radical-directed backbone fragment ions of the a, c, and z varieties. Dotted arrow indicates precursor m/z.

Figure 2

Plots of radical-directed fragmentation as a function of sequence for charge states +4 through +10. Arginine and tyrosine side chain loss intensities are shown as white and black bars, respectively. For each charge state, the sum of RDD backbone fragmentation at each residue and side chain loss intensities are normalized to the highest sum.

Figure 3

Comparison of backbone fragmentation distant from Tyr59 (> 5 residues) summed for each charge state (squares) and the average collisional cross section from ion mobility data (triangles). Fragmentation at distal sites decreases significantly with increasing charge. The inverse relationship with the average collisional cross sections indicates that radical migration to residues that are distant in sequence from Tyr59 diminishes as the protein adopts more elongated structures.

Figure 4

a) CID of [(Ubiquitin)· + 6H]⁶⁺ yields loss of protonated arginine side chain (−87) as the most intense fragment ion. Further MS/MS experiments locate the arginine residue from which the −87 loss originates. b) CID of −87 Da product yields an 86 Da-shifted y fragment ion, which narrows the loss to Arg54, Arg72, and Arg74. CID of the y₂₄ fragment in (b) yields a prominent pseudo b₆ ion containing residues Gly53 through Asp57, which eliminates Arg54 as a candidate (inset). Thus, the side chain loss originates from either Arg72 or Arg74. c) CID of [(Ubiquitin)· +10H]¹⁰⁺ yields loss of tyrosine side chain as most abundant fragment ion. d) Plot of side chain losses from tyrosine and arginine as a function of charge state. ^# neutral loss of H₂O from the molecular ion. † loss of tyrosine sidechain. Dotted arrows indicate precursor m/z.

Figure 5

Selected backbone and side chain fragmentation products as a function of charge state (a–c). Fragmentation at residues 13, 21, and 65 dominate at lower charge states (compact conformers), and disappear by the +8 charge state (a). Partially unfolded conformers are characterized by enhanced fragmentation nearby Tyr59 (b), and fragmentation at residues 54 and 72/74 (c, see text).

Figure 6

a) The crystal structure of ubiquitin with color coding indicating the intensity of RDD backbone fragmentation for the +4 charge state. b) Structure of the +4 charge state of ubiquitin calculated by MDSA using distance constraints derived from experimental data, which are shown as green dotted lines. The coloring is the same as in (a). c) MDSA output without distance constraints. d) The crystal structure of ubiquitin colored by RDD fragment intensities of the +6 charge state. Arg72 and Arg74 are highlighted in red, due to the dominant arginine side chain loss observed. e, f) The output structures of a constrained and unconstrained MDSA calculation, respectively, with identical coloring as (d).