α-Synuclein: Stable compact and extended monomeric structures and pH dependence of dimer formation

  • Summer L. Bernstein
  • Dengfeng Liu
  • Thomas Wyttenbach
  • Michael T. Bowers
  • Jennifer C. Lee
  • Harry B. Gray
  • Jay R. Winkler
Focus: Biological Mass Spectrometry

Abstract

The protein α-synuclein, implicated in Parkinson’s disease, was studied by combining nano-electrospray ionization (N-ESI) mass spectrometry and ion mobility. It was found that both the charge-state distribution in the mass spectra and the average protein shape deduced from ion mobility data, depend on the pH of the spray solution. Negative-ion N-ESI of pH 7 solutions yielded a broad charge-state distribution from −6 to −16, centered at −11, and ion mobility data consistent with extended protein structures. Data obtained for pH 2.5 solutions, on the other hand, showed a narrow charge-state distribution from −6 to −11, centered at −8, and ion mobilities in agreement with compact α-synuclein structures. The data indicated that there are two distinct families of structures: one consisting of relatively compact proteins with eight or less negative charges and one consisting of relatively extended structures with nine or more charges. The average cross section of a-synuclein at pH 2.5 is 33% smaller than for the extended protein sprayed from pH 7 solution. Significant dimer formation was observed when sprayed from pH 7 solution but no dimers were observed from the low pH solution. A plausible mechanism for aggregate formation in solution is proposed.

References

  1. 1.
    Dunnett, S. B.; Bjorklund, A. Prospects for new restorative and neuroprotective treatments in Parkinson’s disease. Nature Suppl. 1999, 399(6738), A32-A39.CrossRefGoogle Scholar
  2. 2.
    Baba, M.; Nakajo, S.; Tu, P. H.; Tomita, T.; Nakaya, K.; Lee, V. M. Y.; Trojanowski, J. Q.; Iwatsubo, T. Aggregation of Alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy Bodies. Am. J. Path. 1998, 152, 879–884.Google Scholar
  3. 3.
    Spillantini, M. G.; Schmidt, M. L.; Lee, V. M. Y.; Trojanowski, J. Q.; Jakes, R.; Goedert, M. Alpha-synuclein in Lewy bodies. Nature 1997, 388, 839–840.CrossRefGoogle Scholar
  4. 4.
    Selkoe, D. J. Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature Suppl. 1999, 399, A23-A31.CrossRefGoogle Scholar
  5. 5.
    Bussell, R.; Eliezer, D. Residual structure and dynamics in Parkinson’s disease-associated mutants of alpha-synuclein. J. Biol. Chem. 2001, 276, 45996–46003.CrossRefGoogle Scholar
  6. 6.
    Serpell, L. C.; Berriman, J.; Jakes, R.; Goedert, M.; Crowther, R. A. Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation. Proc. Natl. Acad. Sci. USA 2000, 97, 4897–4902.CrossRefGoogle Scholar
  7. 7.
    Uversky, V. N.; Li, J.; Fink, A. L. Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein—A possible molecular link between Parkinson’s disease and heavy metal exposure. J. Biol. Chem. 2001, 276, 44284–44296.CrossRefGoogle Scholar
  8. 8.
    Eliezer, D.; Kutluay, E.; Bussell, R.; Brown, G. Conformational properties of alpha-synuclein in its free and lipid-associated states. J. Mol. Biol. 2001, 307, 1061–1073.CrossRefGoogle Scholar
  9. 9.
    Volles, M. J.; Lee, S. J.; Rochet, J. C.; Shtilerman, M. D.; Ding, T. T.; Kessler, J. C.; Lansbury, P. T. Vesicle permeabilization by protofibrillar alpha-synuclein: Implications for the pathogenesis and treatment of Parkinson’s disease. Biochemistry 2001, 40, 7812–7819.CrossRefGoogle Scholar
  10. 10.
    Conway, K. A.; Harper, J. D.; Lansbury, P. T. Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nature Med. 1998, 4, 1318–1320.CrossRefGoogle Scholar
  11. 11.(a)
    Benjamin, D. R.; Robinson, C. V.; Hendrick, J. P.; Hartl, U.; Dobson, C. M. Mass Sepctrometry of Ribosomes and Ribosomal subunits. Proc. Nat. Acad. 1998, 95, 7391–7395.CrossRefGoogle Scholar
  12. 11.(b)
    Rostom, A. A.; Fucini, P.; Benjamin, D. R.; Juenemann, R.; Nierhaus, K. H.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Detection and selective dissociation of intact Ribosomes in a mass spectrometer. Proc. Natl. Acad. Sci. 2000, 97, 5185–5190.CrossRefGoogle Scholar
  13. 12.
    Nettleton, E. J.; Tito, P.; Sunde, M.; Bouchard, M.; Dobson, C. M.; Robinson, C. V. Characterization of the oligomeric states of insulin in self-assembly and amyloid fibril formation by mass spectrometry. J. Biophys. 2000, 79, 1053–1065.CrossRefGoogle Scholar
  14. 13.
    von Helden, G.; Hsu, M. T.; Kemper, P. R.; Bowers, M. T. Structures of carbon cluster ions from 3 to 60 atoms: Linears to rings to fullerenes. J. Chem. Phys. 1991, 95, 3835–3837.CrossRefGoogle Scholar
  15. 14.
    Clemmer, D. E.; Jarrold, M. F. Ion Mobility measurements and their applications to clusters and biomolecules. J. Mass Spectrom. 1997, 92, 577.CrossRefGoogle Scholar
  16. 15.
    Wyttenbach, T.; Bowers, M. T. Gas-Phase conformations: The Ion Mobility/Ion Chromatography method. Top. Curr. Chem. 2003, 225, 207–232.CrossRefGoogle Scholar
  17. 16.
    Wyttenbach, T.; von Helden, G.; Bowers, M. T. Gas-Phase conformation of biological molecules: Bradykinin. J. Am. Chem. Soc. 1996, 118, 8355–8364.CrossRefGoogle Scholar
  18. 17.(a)
    Gidden, J.; Wyttenbach, T.; Jackson, A. T.; Scrivens, J. H.; Bowers, M. T. Gas-phase conformations of synthetic polymers: Poly(ethylene glycol), Poly(propylene glycol), and Poly(tetramethylene glycol). J. Am. Chem. Soc. 2000, 122, 4692–4699.CrossRefGoogle Scholar
  19. 17.(b)
    Gidden, J.; Bowers, M. T. Gas-phase conformations of deprotonated trinucleotides (dGTT, dTGT, and dTTG): The question of zwitterion formation. J. Am. Soc. Mass Spectrom. 2003, 14, 161–170.CrossRefGoogle Scholar
  20. 18.(a)
    Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. Naked protein conformation: Cytochrome c in the gas phase. J. Am. Chem. Soc. 1995, 117, 1014.Google Scholar
  21. 18.(b)
    Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. Protein structure in vacuo: Gas-phase conformations of BPTI and Cytochrome c. J. Am. Chem. Soc. 1997, 119, 2240.CrossRefGoogle Scholar
  22. 18.(c)
    Hudgins, R. R.; Ratner, M. A.; Jarrold, M. F. Design of helices that are stable in vacuo. J. Am. Chem. Soc. 1998, 120, 12974.CrossRefGoogle Scholar
  23. 18.(d)
    Kohtani, M.; Kinnear, B. S.; Jarrold, M. F. Metal-ion enhanced helicity in the gas phase. J. Am. Chem. Soc. 2000, 122, 12377.CrossRefGoogle Scholar
  24. 19.
    Li, J.; Taraszka, J. A.; Counterman, A. E.; Clemmer, D. E. Influence of solvent composition and capillary temperature on the conformations of electrosprayed ions: unfolding of compact ubiquitin conformers from pseudonative and denatured solutions. Int. J. Mass Spectrom. 1999, 185, 37.CrossRefGoogle Scholar
  25. 20.
    Counterman, A. E.; Clemmer, D. E. Large Anhydrous Polyalanine ions: Evidence for extended helices and onset of a more compact state. J. Am. Chem. Soc. 2001, 123, 1490.CrossRefGoogle Scholar
  26. 21.
    Jakes, R.; Spillantini, M. G.; Goedert, M. Identification of 2 distinct synucleins from human brain. FEBS Lett. 1994, 345, 27–32.CrossRefGoogle Scholar
  27. 22.
    Der-Sarkissian, A.; Jao, C. C.; Chen, J.; Langen, R. Structural organization of alpha-synuclein fibrils studied by site-directed spin labeling. J. Biol. Chem. 2003, 37530–37535.Google Scholar
  28. 23.
    Wyttenbach, T.; Kemper, P. R.; Bowers, M. T. Design of a New Electrospray Ion Mobility Mass Spectrometer. Int. J. Mass Spectrom. 2001, 212, 13–23.CrossRefGoogle Scholar
  29. 24.
    Mason, E. A.; McDaniel, E. W. Transport Properties of Ions in Gases. Wiley, New York 1978.Google Scholar
  30. 25.
    Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham, T. E., III.; Wang, J.; Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.; Stanton, R. V.; Cheng, A. L.; Vincent, J. J.; Crowley, M.; Tsui, V.; Radmer, R. J.; Duan, Y.; Pitera, J.; Massova, I.; Seibel, G. L.; Singh, U. C.; Weiner, P. K.; Kollman, P. A. AMBER 7 Unversity of California: San Francisco, 2002.Google Scholar
  31. 26.
    Mathews, C. K.; van Holde, K. E. Biochemistry. Benjamin/Cummings, Redwood City, California; 1990.Google Scholar
  32. 27.
    Shvartsburg, A. A.; Jarrold, M. F. An Exact hard-spheres scattering model for the mobilities of polyatomic ions. Chem Phys Let. 1996, 261, 86–91.CrossRefGoogle Scholar
  33. 28.
    Chowdhury, S. K.; Katta, V.; Chait, B. T. Probing conformational changes in proteins by mass spectromery. J. Am. Chem. Soc. 1990, 112, 9012–9013.CrossRefGoogle Scholar
  34. 29.
    Jarrold, M. F. Peptides and proteins in the vapor phase. Annu. Rev. Phys. Chem. 2000, 51, 179–207.CrossRefGoogle Scholar
  35. 30.
    Uversky, V. N.; Gillespie, J. R.; Fink, A. L. Why are “Natively unfolded” proteins unstructured under physiologic conditions?. Proteins: Struct, Funct, and Genetic. 2000, 41, 415–427.CrossRefGoogle Scholar
  36. 31.
    Uversky, V. N.; Li, J.; Fink, A. L. Evidence of partially folded intermediate in a-synuclein fibril formation. J. Biol. Chem. 2001, 276, 10737–10744.CrossRefGoogle Scholar
  37. 32.
    von Helden, G.; Gotts, N. G.; Bowers, M. T. Experimental evidence for the formation of Fullerenes by collisional heating of carbon rings in the gas phase. Nature 1993, 363, 60–63.CrossRefGoogle Scholar
  38. 33.
    Last, A. M.; Robinson, C. V. Protein folding and interactions revealed by Mass Spectrometry. Curr. Opin. Chem. Biol. 1999, 3, 564–570.CrossRefGoogle Scholar
  39. 34.
    Wyttenbach, T.; von Helden, G.; Bowers, M. T. Conformations of alkali ion cationized Polyethers in the gas phase: Polyethylene Glycol and Bis[(benzo-15-crown-5)-15-ylmethyl Pimelate. Int. J. Mass Spectrom. Ion Proc. 1997, 165, 377.CrossRefGoogle Scholar
  40. 34.
    Gidden, J.; Bushnell, J. E.; Bowers, M. T. Gas-Phase Conformations and Folding Energetics of Oligonucleotides: dTG and dGT. J. Am. Chem. Soc. 2001, 123, 5610–5611.CrossRefGoogle Scholar
  41. 35.
    Bernstein, S. L.; Wyttenbach, T.; Baumketner, A.; Shea J.-E.; Bitan, G.; Teplow, D. T.; Bowers, M. T. Amyloid β-protein: Monomer structure and early aggregation states of Aβ42 and its Pro19 alloform. J. Am. Chem. Soc. (submitted).Google Scholar
  42. 36.
    Gidden, J.; Ferzoco, A.; Baker, E. S.; Bowers, M. T. Duplex formation and the onset of helicity in dCG oligonucleotides in a solvent-free environment. J. Am. Chem. Soc. (in press).Google Scholar
  43. 37.
    Gidden, J.; Baker, E. S.; Ferzoco, A.; Bowers, M. T. Structural motifs of DNA complexes in the gas phase. Int. J. Mass Spectrom. (in press).Google Scholar
  44. 38.
    Baker, E. S.; Bernstein, S. L.; Bowers, M. T. (mss in preparation).Google Scholar
  45. 39.
    Rostam, A. A.; Robinson, C. V. Detection of Intact GroEL chaperonin assembly by Mass Spectrometry. J. Am. Soc. 1999, 121, 4718–4719.CrossRefGoogle Scholar
  46. 40.
    Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ oligomerization through distinct pathways. Proc. Natl. Acad. Sci. USA 2003, 100(1), 330–335.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2004

Authors and Affiliations

  • Summer L. Bernstein
    • 1
  • Dengfeng Liu
    • 1
  • Thomas Wyttenbach
    • 1
  • Michael T. Bowers
    • 1
  • Jennifer C. Lee
    • 2
  • Harry B. Gray
    • 2
  • Jay R. Winkler
    • 2
  1. 1.Department of Chemistry and BiochemistryUniversity of California at Santa BarbaraSanta BarbaraUSA
  2. 2.Beckman InstituteCalifornia Institute of TechnologyPasadenaUSA

Personalised recommendations