Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers

Demmers, Jeroen

doi:10.1073/pnas.050444797

Cited by 63 publications

(89 citation statements)

References 18 publications

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“…This is not surprising because the concentration of DMPC and its hydrophobicity are considerably greater than those of gramicidin. This observation is similar to that made for trans membrane peptides by Heck et al [13]. We were unable to see the dimer of gramicidin, however, in the mass spectrum because the experiment required a low pH quench as well as high temperature and high voltage ESI conditions to break apart the vesicles.…”

Section: Resultssupporting

confidence: 88%

The gramicidin dimer shows both EX1 and EX2 mechanisms of H/D exchange

Chitta

Rempel

Gross

2009

J. Am. Soc. Mass Spectrom.

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We describe the use of H/D amide exchange and electrospray ionization mass spectrometry to study, in organic solvents, the pentadecapeptide gramicidin as a model for protein self association. In methanol-OD, all active H's in the peptide exchange for D within 5 min, indicating a monomer/dimer equilibrium that is shifted towards the fast-exchanging monomer. H/D exchange in n-propanol-OD, however, showed a partially protected gramicidin that slowly converts to a second species that exchanges nearly all the active hydrogens, indicating EX1 kinetics for the H/D exchange. We propose that this behavior is the result of slower rate of unfolding in n-propanol compared to that in methanol. The rate constant for the unfolding of the dimer is the rate of disappearance of the partially protected species, and it agrees within a factor of two with a value reported in literature. The rate constant of dimer refolding can be determined from the ratio of the rate constant for unfolding and the affinity constant for the dimer, which we determined in an earlier study. The unfolding activation energy is 20 kcal mol -1 , determined by performing the exchange experiments as a function of temperature. To study gramicidin in an even more hydrophobic medium than n-propanol, we measured its H/D exchange kinetics in a phospholipids vesicle and found a different H/D amide exchange behavior. Gramicidin is an unusual peptide dimer that can exhibit both EX1 and EX2 mechanisms for its H/D exchange, depending on the solvent. Chitta et al.

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Section: Resultssupporting

confidence: 88%

The gramicidin dimer shows both EX1 and EX2 mechanisms of H/D exchange

Chitta

Rempel

Gross

2009

J. Am. Soc. Mass Spectrom.

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“…The exchange kinetics of amides that are potentially involved in intramolecular H-bonding report local and transient unfolding of secondary structure 30,33,51,52 . MD simulations provide insights into the H-bond dynamics along the backbone as well as into solvation 34,44 .…”

Section: Resultsmentioning

confidence: 99%

The Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix Provides a Rationale for the Sequential Cleavage Mechanism of γ-Secretase

et al. 2013

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The etiology of Alzheimer’s disease depends on the relative abundance of different amyloid-β (Aβ) peptide species. These peptides are produced by sequential proteolytic cleavage within the transmembrane helix of the 99 residue C-terminal fragment of the amyloid precursor protein (C99) by the intramembrane protease γ-secretase. Intramembrane proteolysis is thought to require local unfolding of the substrate helix, which has been proposed to be cleaved as a homodimer. Here, we investigated the backbone dynamics of the substrate helix. Amide exchange experiments of monomeric recombinant C99 and of synthetic transmembrane domain peptides reveal that the N-terminal Gly-rich homodimerization domain exchanges much faster than the C-terminal cleavage region. MD simulations corroborate the differential backbone dynamics, indicate a bending motion at a di-glycine motif connecting dimerization and cleavage regions, and detect significantly different H-bond stabilities at the initial cleavage sites. Our results are consistent with the following hypotheses about cleavage of the substrate. First, the GlyGly hinge may precisely position the substrate within γ-secretase such that its catalytic center must start proteolysis at the known initial cleavage sites. Second, the ratio of cleavage products formed by subsequent sequential proteolysis could be influenced by differential extents of solvation and by the stabilities of H-bonds at alternate initial sites. Third, the flexibility of the Gly-rich domain may facilitate substrate movement within the enzyme during sequential proteolysis. Fourth, dimerization may affect substrate processing by decreasing the dynamics of the dimerization region and by increasing that of the C-terminal part of the cleavage region.

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“…Nonetheless, it is consistent with other experimental techniques; when individual residues are not resolved, multistep kinetics are observed. 18, 24, 51–52 In transmembrane domain proteins, the overall deuterium content has been fit to a triexponential function where the first class of hydrogen atoms exchange rapidly (less than 1 min), the second class exchange slower (on the order of tens of minutes), and the third class virtually never exchanges. 18, 51 Ovispirin is not a transmembrane peptide and it lies mostly parallel to the membrane surface; however, multiple classes of hydrogen atoms likely exist for this mostly α-helical surface-bound peptide.…”

Section: Discussionmentioning

confidence: 99%

“…18, 24, 51–52 In transmembrane domain proteins, the overall deuterium content has been fit to a triexponential function where the first class of hydrogen atoms exchange rapidly (less than 1 min), the second class exchange slower (on the order of tens of minutes), and the third class virtually never exchanges. 18, 51 Ovispirin is not a transmembrane peptide and it lies mostly parallel to the membrane surface; however, multiple classes of hydrogen atoms likely exist for this mostly α-helical surface-bound peptide. The residues that lie completely outside the with membrane will likely have the fastest exchange, residues in the most ordered secondary structure deep in the membrane will likely have the slowest exchange, and then residues somewhat exposed to solvent or in a less ordered secondary structure will have intermediate exchange rates.…”

Section: Discussionmentioning

confidence: 99%

2D IR Cross Peaks Reveal Hydrogen–Deuterium Exchange with Single Residue Specificity

2013

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A form of chemical exchange, hydrogen-deuterium exchange (HDX), has long been used as a method for studying the secondary and tertiary structure of peptides and proteins using mass spectrometry and NMR spectroscopy. Using 2D IR (two dimensional infrared) spectroscopy, we resolve cross peaks between the amide II band and a 13C18O isotope labeled amide I band, which we show measures HDX with site-specific resolution. By rapidly scanning 2D IR spectra using mid-IR pulse shaping, we monitor the kinetics of HDX exchange on-the-fly. For the antimicrobial peptide, ovispirin, bound to membrane bilayers, we find that the amide II peak decays with a biexponential with rate constants of 0.54 ± 0.02 and 0.12 ± 0.01 min−1, which is a measure of the overall HDX in the peptide. The cross peaks between Ile-10 labeled ovispirin and the amide II mode, which specifically monitor HDX kinetics at Ile-10, decay with a single rate constant of 0.36 ± 0.1 min−1. Comparing this exchange rate to theoretically determined exchange rates of Ile-10 for ovispirin in a solution random coil configuration, the exchange rate at Ile-10 is at least 100 times slower, consistent with the known α-helix structure of ovispirin in bilayers. Because backbone isotope labels produce only a very small shift of the amide II band, site-specific HDX cannot be measured with FTIR spectroscopy, which is why 2D IR spectroscopy is needed for these measurements.

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Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers

Cited by 63 publications

References 18 publications

The gramicidin dimer shows both EX1 and EX2 mechanisms of H/D exchange

The gramicidin dimer shows both EX1 and EX2 mechanisms of H/D exchange

The Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix Provides a Rationale for the Sequential Cleavage Mechanism of γ-Secretase

2D IR Cross Peaks Reveal Hydrogen–Deuterium Exchange with Single Residue Specificity

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