Transitions between Elongated Conformations of Ubiquitin [M+11H]¹¹⁺ Enhance Hydrogen/Deuterium Exchange

’ INTRODUCTION Since the development of electrospray ionization for mass spectrometry (MS), several MS-based approaches have been developed for characterizing structures of gas-phase protein ions. These can be categorized as either chemical or physicalmethods. Chemical methods include: ion ion and ion molecule reactions, detection of noncovalent adducts, 9 and monitoring of hydrogen/deuterium (H/D) exchange 12 upon exposure of ions to deuterated solvents. Physical methods include: monitoring products of dissociation, induced by collsionalor photoexcitation or electron capture; 17 determination of ion shapes by ion mobility spectrometry (IMS) measurements; and microscopy studies of hillocks formed upon ions hitting surfaces at high energies. Although structural informationwith atomic precision is not accessible, information from different methods provides insight about the number of favored states, their overall shapes, and pathways connecting them. In the present work, we use a combination of IMS and H/D exchange measurements to study structural transitions of [M þ 11H]11þ ions of bovine ubiquitin. The mobilities of ions depend upon their collision cross sections. Thus, IMSmeasurements can provide direct information about an ion’s three-dimensional shape. Relatively small proteins, such as ubiqutin, cytochrome c, lysozyme, and apomyoglobin, often unfold with increasing charge state. The elongated states that persist over extended time periods (milliseconds to seconds) in the gas phase are favored because such structures minimize repulsive columbic interactions. Recently, IMS IMS instrumentation has been developed specifically to study structural transitions. With this approach, a mixture of conformations can be separated in an initial drift region.A narrow range of conformers having a defined mobility are then selected and exposed to energizing collisions so that new populations of states are produced. These new structures are subsequently separated in a second IMS drift region. Such an approach makes it possible to follow folding and unfolding transitions in some detail. We focus on ubiquitin in this work because it is a relatively small model system that has been the subject of numerous experimental studies, including H/D exchange, 33 IMS and IMS IMS, 38 and dissociation studies. Bovine ubiquitin is comprised of 76 amino acid residues and has 144 exchangeable heteroatom hydrogen sites. Its sequence is identical to that of human ubiquitin. The [M þ 11H]11þ charge state favors elongated conformations, having little or no tertiary structure. In their earliest studies, McLafferty and co-workers exposed the ubiquitin ions to 10 7 Torr of D2O for >1000 s by trapping the ions in a Fourier transform (FT) ion cyclotron resonance (ICR) cell. They reported a single value of ∼50 exchangeable sites for [M þ 11H]11þ ubiquitin ions. In 1999, Marshall’s group published a FT-ICR mass spectrum for the [M þ 11H]11þ state of ubiquitin that provided evidence of two ion populations. The largest population of ions exchanged ∼60 sites; a smaller fraction of ions underwent ∼80 exchanges. All of these values for the [M þ 11H]11þ state are far below the maximum number of possible exchanges (144 plus 11 protons); generally, nonreactive sites on elongated states are interpreted as being protected by secondary structural elements through hydrogen bonding. Additionally, the orientation of charged sites on highly extended states may not favor efficient exchange because