Abstract
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 the slower rate of unfolding in n-propanol compared with 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.
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References
Busath, D. D. The Use of Physical Methods in Determining Gramicidin Channel Structure and Function. Ann. Rev. Physiol. 1993, 55, 473–501.
Doyle, D. A.; Wallace, B. A. The Dynamic Nature of Gramicidin. Biomembranes 1996, 6, 327–359.
Wallace, B. A. Recent Advances in the High Resolution Structures of Bacterial Channels: Gramicidin A. J. Struct. Biol. 1998, 121(2), 123–141.
Sarges, R.; Witkop, B.; Gramicidin, A. V. The Structure of Valine- and Isoleucine-Gramicidin A. J. Am. Chem. Soc. 1965, 87(9), 2011–2020.
Ferraro, D. M.; Robertson, A. D. EX1 Hydrogen Exchange and Protein Folding. Biochemistry 2004, 43(3), 587–594.
Deng, Y.; Smith, D. L. Identification of Unfolding Domains in Large Proteins by Their Unfolding Rates. Biochemistry 1998, 37(18), 6256–6262.
Yi, Q.; Baker, D. Direct Evidence for a Two-State Protein Unfolding Transition from Hydrogen-Deuterium Exchange, Mass Spectrometry, and NMR. Prot. Sci. 1996, 5(6), 1060–1066.
Engen, J. R.; Smith, D. L. Investigating Protein Structure and Dynamics by Hydrogen Exchange MS. Anal. Chem. 2001, 73(9), 256A-265A.
Kaltashov, I. A.; Eyles, S. J. Crossing the Phase Boundary to Study Protein Dynamics and Function: Combination of Amide Hydrogen Exchange in Solution and Ion Fragmentation in the Gas Phase. J. Mass Spectrom. 2002, 37(6), 557–565.
Urry, D. W. The Gramicidin A Transmembrane channel: A Proposed pi(L,D) Helix. Proc. Natl. Acad. Sci. U.S.A. 1971, 68(3), 672–676.
Veatch, W. R.; Fossel, E. T.; Blout, E. R. The Conformation of Gramicidin A. Biochemistry 1974, 13(26), 5249–5256.
Akashi, S.; Takio, K. Structure of Melittin Bound to Phospholipid Micelles Studied Using Hydrogen-Deuterium Exchange and Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12(12), 1246–1253.
Demmers, J. A.; Haverkamp, J.; Heck, A. J.; Koeppe, R. E. II; Killian, J. A. Electrospray Ionization Mass Spectrometry as a Tool to Analyze Hydrogen/Deuterium Exchange Kinetics of Transmembrane Peptides in Lipid Bilayers. Proc. Nat. Acad. Sci. U.S.A. 2000, 97(7), 3189–3194.
Demmers, J. A.; Van Duijn, E.; Haverkamp, J.; Greathouse, D. V.; Koeppe, R. E. II; Heck, A. J. R.; Killian, J. A. Interfacial Positioning and Stability of Transmembrane Peptides in Lipid Bilayers Studied by Combining Hydrogen/Deuterium Exchange and Mass Spectrometry. J. Biol. Chem. 2001, 276(37), 34501–34508.
Hansen, R. K.; Broadhurst, R. W.; Skelton, P. C.; Arkin, I. T. Hydrogen/Deuterium Exchange of Hydrophobic Peptides in Model Membranes by Electrospray Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2002, 13(12), 1376–1387.
Sidhu, S. S.; Fairbrother, W. J.; Deshayes, K. Exploring Protein-Protein Interactions with Phage Display. Chem. Biochem. 2003, 4(1), 14–25.
Veenhoff, L. M.; Heuberger, E. H. M. L.; Poolman, B. Quaternary Structure and Function of Transport Proteins. Trends Biochem. Sci. 2002, 27(5), 242–249.
Veselovsky, A. V.; vanov, Y. D.; Ivanov, A. S.; Archakov, A. I.; Lewi, P.; Janssen, P. Protein-Protein Interactions: Mechanisms and Modification by Drugs. J. Mol. Recog. 2002, 5(6), 405–422.
Barone, G.; Giancola, C. Peptide-Peptide Interactions in Water and Concentrated Urea Solutions. Pure Appl. Chemi. 1990, 62(1), 57–68.
Liu, D.; Huang, Z. Synthetic Peptides and Nonpeptidic Molecules as Probes of Structure and Function of Bcl-2 Family Proteins and Modulators of Apoptosis. Apoptosis 2001, 6(6), 453–462.
Xing, G.; DeRose, V. J. Designing Metal-Peptide Models for Protein Structure and Function. Curr. Opin. Chem. Biol. 2001, 5(2), 196–200.
Bechinger, B. Structure and Functions of Channel-Forming Peptides: Magainins, Cecropins, Melittin, and Alamethicin. J. Membr. Biol. 1997, 156(3), 197–211.
Kaiser, E. T.; Kezdy, F. J. Peptides with Affinity for Membranes. Ann. Rev. Biophys., Biophys. Chem. 1987, 16, 561–581.
Chitta, R. K.; Gross, M. L. Electrospray Ionization-Mass Spectrometry and Tandem Mass Spectrometry Reveal Self-Association and Metal-Ion Binding of Hydrophobic Peptides: A Study of the Gramicidin Dimer. Biophys. J. 2004, 86(1), 473–479.
Chitta, R. K.; Rempel, D. L.; Gross, M. L. Determination of Affinity Constants and Response Factors of the Noncovalent Dimer of Gramicidin by Electrospray Ionization Mass Spectrometry and Mathematical Modeling. J. Am. Soc. Mass Spectrom. 2005, 16(7), 1031–1038.
Ehring, H. Hydrogen Exchange/Electrospray Ionization Mass Spectrometry Studies of Structural Features of Proteins and Protein/Protein Interactions. Anal. Biochem. 1999, 267(2), 252–259.
Smith, D. L. Local structure and dynamics in proteins characterized by hydrogen exchange and mass spectrometry. Biochemistry 1998, 63(3), 285–293.
LoGrasso, P. V.; Moll, F. III; Cross, T. A. Solvent History Dependence of Gramicidin A Conformations in Hydrated Lipid Bilayers. Biophys. J. 1988, 54(2), 259–267.
Rockwood, A. L.; Kushnir, M. M.; Nelson, G. J. Dissociation of Individual Isotopic Peaks: Predicting Isotopic Distributions of Product Ions in MSn. J. Am. Soc. Mass Spectrom. 2003, 14(4), 12.
Birnbaum, D. T.; Dodd, S. W.; Saxberg, B. E. H.; Varshavsky, A. D.; Beals, J. M. Hierarchical Modeling of Phenolic Ligand Binding to 2Zn-Insulin Hexamers. Biochemistry 1996, 35(17), 5366–5378.
Knuth, K. H.; Hajian, A. R. Hierarchies of Models: Toward Understanding Planetary Nebulae Proceedings of the American Institute of Physics Conference Moscow, ID, August 2002; 659(1), p. 92.
Braco, L.; Bano, C.; Chillaron, F.; Abad, C. Dimer-Monomer Conformational Equilibrium of Gramicidin A in 1-Alkanols as Studied by HPLC and Fluorescence Spectroscopy. Int. J. Biol. Macromol. 1988, 10(6), 343–348.
Chung, E. W.; Nettleton, E. J.; Morgan, C. J.; Grob, M.; Miranker, A.; Radford, S. E.; Dobson, C. M.; Robinson, C. V. Hydrogen Exchange Properties of Proteins in Native and Denatured States Monitored by Mass Spectrometry and NMR. Prot. Sci. 1997, 6(6), 1316–1324.
Miranker, A.; Robinson, C. V.; Radford, S. E.; Aplin, R. T.; Dobson, C. M. Detection of Transient Protein Folding Populations by Mass Spectrometry. Science (Washington, DC) 1993, 262(5135), 896–900.
Breuker, K. New Mass Spectrometric Methods for the Quantification of Protein-Ligand Binding in Solution. Angew. Chem. Int. Ed. 2004, 43(1), 22–25.
Swint-Kruse, L.; Robertson, A. D. Temperature and pH Dependences of Hydrogen Exchange and Global Stability for Ovomucoid Third Domain. Biochemistry 1996, 35(1), 171–180.
Bamberg, E.; Laeuger, P. Temperature Dependent Properties of Gramicidin A Channels. Biochim. Biophys. Acta 1974, 367(2), 127–133.
Jorgensen, T. J. D.; Cheng, L.; Heegaard, N. H. H. Mass spectrometric characterization of conformational preludes to β2-microglobulin aggregation. Int. J. Mass Spectrom. 2007, 268(2–3), 207–216.
Grasberger, B.; Minton, A. P.; DeLisi, C.; Metzger, H. Interaction Between Proteins Localized in Membranes. Proc. Acad. Sci. U.S.A. 1986, 83(17), 6258–6262.
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Published online June 21, 2009
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Chitta, R.K., Rempel, D.L. & Gross, M.L. The gramicidin dimer shows both EX1 and EX2 mechanisms of H/D exchange. J Am Soc Mass Spectrom 20, 1813–1820 (2009). https://doi.org/10.1016/j.jasms.2009.05.017
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DOI: https://doi.org/10.1016/j.jasms.2009.05.017