Gas-phase folding and unfolding of cytochrome c cations.

109

Isotopic exchange reactions of compact and elongated conformations of gaseous cytochrome c ions (ϩ5 and ϩ9 states) with D 2 O have been measured as a function of temperature (from 300 to ϳ440 K) using ion mobility techniques. Rate constants for those sites that exchange at high temperatures (Ͼ400 K) are about an order of magnitude smaller than rate constants for sites that exchange at 300 K. Although the exchange rates decrease, the maximum exchange levels for rapidly exchanging sites increase with temperature. At 300 K, exchange levels of 53 Ϯ 3 and 63 Ϯ 3 are measured for the compact and elongated states, respectively. From 300 to 335 K, the exchange levels increase slightly to ϳ60 to 70 hydrogens. Above 335 K, the levels increase to a value of ϳ200 for the ϩ5 state and ϳ190 for the ϩ9 state, near the maximum possible levels, 200 and 204 for these respective charge states. Molecular dynamics simulations have been carried out on structures having calculated cross sections that are near the experimental values in order to explore the exchange process. Overall, it appears that charge site and exchange site proximities are important factors in the exchange profiles for the elongated ϩ9 ion and the compact ϩ5 ion. (J Am Soc Mass Spectrom 2002, 13, 506 -517) © 2002 American Society for Mass Spectrometry I t is well known that the structures of proteins are influenced by environmental factors such as solution composition, pH, and temperature. However, the degree to which environment and intrinsic properties influence structure is not well understood. Structural domains such as ␣-helices, which are stable in a wide range of environments, were predicted theoretically without considering effects of solvation [1]. More subtle features, such as tertiary structure in different environments, are difficult to predict. Recently, efforts have been made to gather information about the degree to which solvent-free protein conformations resemble their native solution structures [2]. Several studies of lyophilized protein powders have shown that removal of solvent can cause structural changes [3], and the extent of differences between lyophilized and solution states is an active area [4]. New ion sources [5,6] make it possible to isolate biomolecules in the gas phase as ions, and a number of mass spectrometry (MS) based strategies for examining solvent-free conformations are being developed . The new information from these technologies is beginning to be evaluated by theoretical methods [33, 34,35]. There is now substantial evidence that, under some experimental conditions, anhydrous protein and peptide ions may retain elements of solution conformation.The conformations of cytochrome c ions have been examined previously by a number of MS techniques [8, 28]. Ion mobility (for reviews of ion mobility studies see [30]) and isotopic exchange studies [7-10, 11, 12] provide information about the overall shape and number of accessible exchange sites, respectively. These studies indicate the presence of multiple conformations within ind...

Section: H/d Exchange Kineticssupporting

confidence: 92%

mentioning

confidence: 99%

Temperature-dependent H/D exchange of compact and elongated cytochrome c ions in the gas phase

Valentine

Clemmer

2002

109

“…A number of different methods can provide information about molecular structure in the gas phase, even complex systems, such as large synthetic or biological polymers. These methods include spectroscopy [1][2][3][4][5], hydrogen/deuterium exchange [6][7][8][9][10][11][12], dissociation [13][14][15][16][17][18], proton transfer reactivity [19][20][21][22], and ion mobility mass spectrometry . These methods have been used to obtain information about protein conformation and folding, DNA duplex structure, and in a wide variety of other interesting applications.…”

mentioning

confidence: 99%

Evaluation of ion mobility spectroscopy for determining charge-solvated versus salt-bridge structures of protonated trimers

Wong

Williams

Counterman

et al. 2005

The cross sections of five different protonated trimers consisting of two base molecules and trifluoroacetic acid were measured by using ion mobility spectrometry. The gas-phase basicities of these five base molecules span an 8-kcal/mol range. These cross sections are compared with those determined from candidate low-energy salt-bridge and charge-solvated structures identified by using molecular mechanics calculations using three different force fields: AMBER*, MMFF, and CHARMm. With AMBER*, the charge-solvated structures are all globular and the salt-bridge structures are all linear, whereas with CHARMm, these two forms of the protonated trimers can adopt either shape. Globular structures have smaller cross sections than linear structures. Conclusions about the structure of these protonated trimers are highly dependent on the force field used to generate low-energy candidate structures. With AMBER*, all of the trimers are consistent with salt-bridge structures, whereas with MMFF the measured cross sections are more consistent with charge-solvated structures, although the assignments are ambiguous for two of the protonated trimers. Conclusions based on structures generated by using CHARMm suggest a change in structure from charge-solvated to salt-bridge structures with increasing gas-phase basicity of the constituent bases, a result that is most consistent with structural conclusions based on blackbody infrared radiative dissociation experiments for these protonated trimers and theoretical calculations on the uncharged base-acid pairs. T he structure of a molecule in solution depends both on intramolecular interactions and on intermolecular interactions between the molecule and solvent. Gas-phase studies can provide information about the intrinsic properties of a molecule. From such measurements, information about solvent effects can be inferred. A number of different methods can provide information about molecular structure in the gas phase, even complex systems, such as large synthetic or biological polymers. These methods include spectroscopy [1][2][3][4][5], hydrogen/deuterium exchange [6-12], dissociation [13][14][15][16][17][18], proton transfer reactivity [19][20][21][22], and ion mobility mass spectrometry . These methods have been used to obtain information about protein conformation and folding, DNA duplex structure, and in a wide variety of other interesting applications.Of these methods, ion mobility mass spectrometry, a method pioneered by Bowers and Jarrold for ion structure characterization, has several advantages, including rapid measurement time, minimal perturbation of ground state structure, exquisite temperature control, and the ability to investigate kinetics of structural change over a limited time. With ion mobility, ions are injected into a drift tube where they thermalize through collisions with an introduced background gas, typically helium. In the presence of an applied electric field, ions are made to drift through an elevated pressure region. Based on the drift time through this tube...

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confidence: 99%

“…The gentleness of ESI allows H/D exchange experiments in the gas phase to give information about protein conformation and protein/protein interactions based on measurements of the exchange rates of protein backbone amide protons [8,9]. This combination of delicate handling of sensitive biological macromolecules and high resolution mass spectrometry now has the potential to yield insight into ongoing biological activity [10,11].…”

mentioning

confidence: 99%

Fourier transform-ion cyclotron resonance mass spectrometric resolution, identification, and screening of non-covalent complexes of Hck Src homology 2 domain receptor and ligands from a 324-member peptide combinatorial library

Wigger

Eyler

Benner

et al. 2002

The preferred ligands for the Hck Src homology 2 domain among a combinatorial library containing 324 different peptides were determined in a single experiment involving Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), electrospray ionization (ESI), stored-waveform inverse Fourier transformation (SWIFT), and infrared multiphoton laser disassociation (IRMPD). These were compared with the results obtained by conventional screening of the peptide library in solution using affinity chromatography. The results reported here show that by combining ESI, FT-ICR MS, SWIFT, and IRMPD, ligands likely to bind under physiological conditions are rapidly and efficiently identified, even from complex library mixtures. In the gas phase some discrimination against hydrophobic ligands could be observed. However, the illustrated feasibility of identifying high affinity ligand via gas-phase screening of complex library mixtures should lead to broad applications in the development of ligands for proteins with interesting biological activity, the first step that must be taken to develop a therapeutic agent. Important in the application of FT-ICR methods to biological macromolecules are "soft" ionization techniques, which generate macromolecular ions without fragmentation. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are two of these [4,5]. ESI involves spraying an aqueous solution of a biological macromolecule under "quasiphysiological" conditions directly into a mass spectrometer. The solvent evaporates in the vacuum from the droplets, gently "elevating" a biological ion from its native environment in solution. This process frequently leaves non-covalent macromolecular complexes intact and allows the mass spectrometric observation of enzyme-substrate, receptor-ligand, peptide-peptide, protein-subunit, oligonucleotide-toxin/drug, and DNA duplex binding in the gas phase [6].In many cases, binding constants estimated from mass spectrometric studies match the binding constants obtained in solution to within an order of magnitude [7]. The gentleness of ESI allows H/D exchange experiments in the gas phase to give information about protein conformation and protein/protein interactions based on measurements of the exchange rates of protein backbone amide protons [8,9]. This combination of