Self-Assembly of Protein Fibrils in Microgravity

Dylan Bell, Samuel Durrance, Daniel Kirk, Hector Gutierrez, Daniel Woodard, Jose Avendano, Joseph Sargent, Caroline Leite, Beatriz Saldana, Tucker Melles, Samantha Jackson, Shaohua Xu

Abstract


Deposits of insoluble protein fibrils in human tissue are associated with amyloidosis and neurodegenerative diseases. Different proteins are involved in each disease; all are soluble in their native conformation in vivo, but by molecular self-assembly, they all form insoluble protein fibril deposits with a similar cross β-sheet structure. This paper reports the results of an experiment in molecular self-assembly carried out in microgravity on the International Space Station (ISS). The Self-Assembly in Biology and the Origin of Life (SABOL) experiment was designed to study the growth of lysozyme fibrils in microgravity. Lysozyme is a model protein that has been shown to replicate the aggregation processes of other amyloid proteins. Here the design and performance of the experimental hardware is described in detail. The flight experiment was carried to the ISS in the Dragon capsule of the SpaceX CRS-5 mission and returned to Earth after 32 days. The lysozyme fibrils formed in microgravity aboard the ISS show a distinctly different morphology compared to fibrils formed in the ground-control (G-C) experiment. The fibrils formed in microgravity are shorter, straighter, and thicker than those formed in the laboratory G-C experiment. For two incubation periods, (2) about 8.5 days and (3) about 14.5 days, the average ISS and G-C fibril diameters are respectively:

Period 2 DISS = 7.5nm ± 31%,
and DG-C = 3.4nm ± 31%

Period 3 DISS = 6.2nm ± 33%,
and DG-C = 3.6nm ± 33%.

References


Aggeli A, Boden N (2006) Self-assembling peptide gels. In Molecular Gels: Materials with Self-assembled Fibrillar Networks, pp 99-130. Dordrecht: Springer

Bitan G, Fradinger EA, Spring SM, Teplow DB (2005) Neurotoxic protein oligomers: what you see is not always what you get. Amyloid 12(2): 88–95

Booth DR, Sunde M, Bellotti V, Robinson CV, Hutchinson WL, Fraser PE, Hawkins PN, Dobson CM, Radford SE, Blake CCF, Pepys MB (1997) Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 385(6619): 787

Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, Stefani M (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416(6880): 507–511

Burnett LC, Burnett BJ, Li B, Durrance ST, Xu S (2014) A lysozyme concentration, pH, and time-dependent isothermal transformation diagram reveals fibrous amyloid and non-fibrous, amorphous aggregate species. Open Journal of Biophysics 4: 39-50

Canet D, Sunde M, Last AM, Miranker A, Spencer A, Robinson CV, Dobson CM (1999) Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants. Biochemistry 38(20): 6419–6427

Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Annual Review of Biochemistry 75: 333–366

Chiti F, Webster P, Taddei N, Clark A, Stefani M, Ramponi G, Dobson CM (1999) Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proceedings of the National Academy of Sciences USA 96(7): 3590–3594

Dahlgren KN, Manelli AM, Stine WB, Baker LK, Krafft GA, LaDu MJ (2002) Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability. Journal of Biological Chemistry 277(35): 32046–32053

Dobson CM (2004) In the footsteps of alchemists. Science 304(5675): 1259–1262

Estroff LA, Hamilton AD (2006) Cryo-tem, x-ray diffraction and modeling of an organic hydrogel. In Molecular Gels: Materials with Self-assembled Fibrillar Networks, pp 721–742. Dordrecht: Springer

Frare E, Mossuto MF, de Laureto PP, Dumoulin M, Dobson CM, Fontana A (2006) Identification of the core structure of lysozyme amyloid fibrils by proteolysis. Journal of Molecular Biology 361(3): 551–561

Fujiwara S, Matsumoto F, Yonezawa Y (2003) Effects of salt concentration on association of the amyloid protofilaments of hen egg white lysozyme studied by time-resolved neutron scattering. Journal of Molecular Biology 331(1): 21–28

Guijarro JI, Sunde M, Jones JA, Campbell ID, Dobson CM (1998) Amyloid fibril formation by an sh3 domain. Proceedings of the National Academy of Sciences USA 95(8):4224– 4228

Hill SE, Miti T, Richmond T, Muschol M (2011). Spatial extent of charge repulsion regulates assembly pathways for lysozyme amyloid fibrils. PLOS One 6(4): e18171

Hill SE, Robinson J, Matthews G, Muschol M (2009) Amyloid protofibrils of lysozyme nucleate and grow via oligomer fusion. Biophysical Journal 96(9): 3781–3790

Jim´enez JL, Nettleton EJ, Bouchard M, Robinson CV, Dobson CM, Saibil HR (2002) The protofilament structure of insulin amyloid fibrils. Proceedings of the National Academy of Sciences USA 99(14): 9196–9201

Kallberg Y, Gustafsson M, Persson B, Thyberg J, Johansson J (2001) Prediction of amyloid fibril-forming proteins. Journal of Biological Chemistry 276(16): 12945–12950

Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300(5618): 486–489

Kelly JW (1996) Alternative conformations of amyloidogenic proteins govern their behavior. Current Opinion in Structural Biology 6(1): 11–17

Kodali R, Wetzel R (2007) Polymorphism in the intermediates and products of amyloid assembly. Current Opinion in Structural Biology 17(1): 48–57

Kowalewski T, Holtzman DM (1999) In situ atomic force microscopy study of Alzheimer’s β-amyloid peptide on different substrates: New insights into mechanism of β-sheet formation. Proceedings of the National Academy of Sciences USA 96(7): 3688–3693

Lansbury PT, Lashuel HA (2006) A century-old debate on protein aggregation and neurodegeneration enters the clinic. Nature 443(7113): 774–779

Necula M, Kayed R, Milton S, Glabe CG (2007) Small molecule inhibitors of aggregation indicate that amyloid β oligomerization and fibrillization pathways are independent and distinct. Journal of Biological Chemistry 282(14): 10311–10324

Pellarin R, Caflisch A (2006) Interpreting the aggregation kinetics of amyloid peptides. Journal of Molecular Biology 360(4):882–892

Pepys MB, Hawkins PN, Booth DR, Vigushin DM, Tennent GA, Soutar AK, Totty N, Nguyen O, Blake CCF, Terry CJ, Feest TG, Zalin AM, Hsuan JJ (1993). Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature 362(6420): 553

Perutz M, Finch J, Berriman J, Lesk A (2002) Amyloid fibers are water-filled nanotubes. Proceedings of the National Academy of Sciences USA 99(8): 5591–5595

Ross CA, Poirier MA (2004) Protein aggregation and neurodegenerative disease. Nature Medicine 10(7): S10

Sunde M, Blake C (1997) The structure of amyloid fibrils by electron microscopy and x-ray diffraction. Advances in Protein Chemistry 50: 123–159

Tabony J, Rigotti N, Glade N, Cort`es S (2007) Effect of weightlessness on colloidal particle transport and segregation in self-organising microtubule preparations. Biophysical Chemistry 127(3):172–180

Terech P (2006) Gels. In Encyclopedia of Surface and Colloid Science, 2nd ed, vol 4, pp 2678–2696. London: Taylor and Francis

Wang F, Hayter J, Wilson LJ (1996) Salt-induced aggregation of lysozyme studied by cross-linking with glutaraldehyde: implications for crystal growth. Acta Crystallographica. Section D, Biological Crystallography 52(Pt 5): 901–908

Ward S, Himmelstein D, Lancia J, Binder L (2012) Tau oligomers and tau toxicity in neurodegenerative disease. Biochemical Society Transactions 40(4): 667

Woodard D, Bell D, Tipton D, Durrance S, Cole L, Li B, Xu S (2014) Gel formation in protein amyloid aggregation: a physical mechanism for cytotoxicity. PLOS One 9(4): e94789


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