Preparation of α-synuclein fibrils for solid-state NMR: Expression, purification, and incubation of wild-type and mutant forms
作者： Kathryn D Kloepper , Wendy S Woods , Kem A Winter , Julia M George , Chad M Rienstra
DOI: 10.1016/J.PEP.2006.02.009
关键词:
摘要: Abstract We report the expression and purification of α-synuclein, a protein implicated in Parkinson’s disease, from isotopically ( 13 C, 15 N) labeled bacterial growth media, as required for solid-state NMR structural studies. Expression Escherichia coli (BL21(DE3)) was performed with protocol optimized time efficiency yield. Chemical lysis, crude by ammonium sulfate precipitation, two chromatography steps (hydrophobic interaction size exclusion) yield 30–35 mg/L medium. Purity is confirmed gel electrophoresis mass spectrometry. Furthermore, we demonstrate reproducible fibril control environmental incubation conditions. Highly resolved multidimensional spectra indicate microscopic order throughout majority AS structure. The number signals intensities well-resolved residue types (Thr, Ser, Ala, Gly, Val, Ile) are consistent single conformation, which reproducibly prepared seeding consecutive preparations. Variations rates polymorphisms exhibited minimized careful
elsevier.com
sci-hub.se 参考文章(25)
Terra C. Holdeman, Kevin H. Gardner, 1H, 13C and 15N chemical shift assignments of the N-terminal PAS domain of mNPAS2 [1] Journal of Biomolecular NMR. ,vol. 21, pp. 383- 384 ,(2001) , 10.1023/A:1013334608913
Maria Grazia Spillantini, Marie Luise Schmidt, Virginia M.-Y. Lee, John Q. Trojanowski, Ross Jakes, Michel Goedert, α-Synuclein in Lewy bodies Nature. ,vol. 388, pp. 839- 840 ,(1997) , 10.1038/42166
Juan J. Zarranz, Javier Alegre, Juan C. Gómez-Esteban, Elena Lezcano, Raquel Ros, Israel Ampuero, Lídice Vidal, Janet Hoenicka, Olga Rodriguez, Begoña Atarés, Verónica Llorens, Estrella Gomez Tortosa, Teodoro del Ser, David G. Muñoz, Justo G. de Yebenes, The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Annals of Neurology. ,vol. 55, pp. 164- 173 ,(2004) , 10.1002/ANA.10795
T. L. S. Benzinger, D. M. Gregory, T. S. Burkoth, H. Miller-Auer, D. G. Lynn, R. E. Botto, S. C. Meredith, Propagating structure of Alzheimer’s β-amyloid(10–35) is parallel β-sheet with residues in exact register Proceedings of the National Academy of Sciences of the United States of America. ,vol. 95, pp. 13407- 13412 ,(1998) , 10.1073/PNAS.95.23.13407
Robert Tycko, Progress towards a molecular-level structural understanding of amyloid fibrils. Current Opinion in Structural Biology. ,vol. 14, pp. 96- 103 ,(2004) , 10.1016/J.SBI.2003.12.002
Robert Tycko, Applications of solid state NMR to the structural characterization of amyloid fibrils: methods and results Progress in Nuclear Magnetic Resonance Spectroscopy. ,vol. 42, pp. 53- 68 ,(2003) , 10.1016/S0079-6565(03)00003-7
Janet M. Griffiths, Ted T. Ashburn, Michele Auger, Philip R. Costa, Robert G. Griffin, Peter T. Lansbury, Rotational Resonance Solid-State NMR Elucidates a Structural Model of Pancreatic Amyloid Journal of the American Chemical Society. ,vol. 117, pp. 3539- 3546 ,(1995) , 10.1021/JA00117A023
Kelly A. Conway, James D. Harper, Peter T. Lansbury, Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nature Medicine. ,vol. 4, pp. 1318- 1320 ,(1998) , 10.1038/3311
H. Heise, W. Hoyer, S. Becker, O. C. Andronesi, D. Riedel, M. Baldus, Molecular-level secondary structure, polymorphism, and dynamics of full-length ¿-synuclein fibrils studied by solid-state NMR Proceedings of the National Academy of Sciences of the United States of America. ,vol. 102, pp. 15871- 15876 ,(2005) , 10.1073/PNAS.0506109102
Seung R. PAIK, Hyun-Ju SHIN, Ju-Hyun LEE, Chung-Soon CHANG, Jongsun KIM, Copper(II)-induced self-oligomerization of alpha-synuclein. Biochemical Journal. ,vol. 340, pp. 821- 828 ,(1999) , 10.1042/BJ3400821