pubs.acs.org/Langmuir © 2009 American Chemical Society
Amyloid Gels: Precocious Appearance of Elastic Properties during the Formation of an Insulin Fibrillar Network Mauro Manno,*,† Daniela Giacomazza,† Jay Newman,‡ Vincenzo Martorana,† and Pier Luigi San Biagio† †
Institute of Biophysics at Palermo, Italian National Research Council, via Ugo La Malfa 153, I90146 Palermo, Italy, and ‡Physics and Astronomy Department, Union College, Schenectady, New York 12308 Received September 4, 2009. Revised Manuscript Received November 5, 2009
The formation of insulin amyloid fibrils is important not only for the development of reliable drugs but also for modeling the basic properties of protein self-assembly. Fibrillation kinetics is typically characterized by an initial apparent lag phase related to the formation of oligomers, protofibrils, and aggregation nuclei. Afterwards, aggregation proceeds over a wide range of length scales via fibril elongation, thickening, and/or flocculation and eventual gelation. By light scattering and rheological techniques, we reveal the structural details hidden in the apparent lag phase and we show the unexpected appearance of noteworthy elastic properties concurrently with initial fibril nucleation and elongation preceding the formation of the larger structures and the gel network.
Amyloid fibrils are insoluble linearly elongated protein aggregates exhibiting a typical cross-beta structure. They play a pivotal role in many pathologies, including several neurodegenerative diseases, as well as in other clinically relevant issues.1 A less-wellknown aspect of amyloid fibrils is the ubiquitous presence of positive functional amyloids.1,2 This observation suggests a comparison with other known protein filamentous networks that have a well-known physiological function, such as the cytoskeleton proteins.1 Also, the new capabilities of bioengineering create new possibilities for controlling the structural and mechanical properties of protein or peptide fibrillar networks.4 These advancements foster opportunities for using protein or peptide fibrillar assembly, including amyloid-like fibrils, as new biomolecular materials,5 with a potential application in tissue engineering as a cell scaffold.6 The outlined scenario strengthens the need for further studies on the basic aspects of both the structural and mechanical properties of amyloid fibrillar networks.7 Here, we address such a topic in the case of insulin fibrillation. Our experiments disclose the unexpected and in some respects counterintuitive appearance of elastic solidlike behavior preceding the actual formation of extensive network-forming structures. We also revealed the formation of fibril nuclei or precursors during the apparent lag phase. We study the formation of amyloid fibrillar aggregates of bovine insulin and their mechanical and structural properties, up to the eventual appearance of gel-like behavior, by using timeresolved light-scattering and rheological experiments. The formation of insulin aggregates is enriched by a complex hierarchy of *Corresponding author. Tel: þ39-091-690305. Fax: þ39-091-6809349. E-mail:
[email protected]. (1) Chiti, F.; Dobson, C. M. Annu. Rev. Biochem. 2006, 75, 333366. (2) Otzen, D. E.; Nielsen, P. H. Cell. Mol. Life Sci. 2008, 65, 910927. (4) Aggeli, A.; Nyrkova, I. A.; Bell, M.; Harding, R.; Carrick, L.; McLeish, T. C. B.; Semenov, A. N.; Boden, N. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 11857– 11862. (5) Lashuel, H. A.; LaBrenz, S. R.; Woo, L.; Serpell, L. C.; Kelly, J. W. J. Am. Chem. Soc. 2000, 122, 5262–5277. (6) Eyrich, D.; Brand, F.; Appel, B.; Wiese, H.; Maier, G.; Wenzel, M.; Staudenmaier, R.; Goepferich, A.; Blunk, T. Biomaterials 2007, 28, 5565. (7) Storm, C.; Pastore, J. J.; MacKintosh, F. C.; Lubensky, T. C.; Janmey, P. A. Nature 2005, 435, 191–194.
1424 DOI: 10.1021/la903340v
different morphologies and by the concurrent operation of different processes.8-10 The first step in the aggregation process is the so-called lag phase, which is related to the formation of oligomeric active centers (nucleation)8 or to the first assembly of oligomers and protofibrils.11,12 However, the lag phase is mainly due to a lack of experimental sensitivity, and its features remain mostly unknown.13 The second step is related to an autocatalytic (“downhill”) mechanism, which results in fibril elongation14 or the formation of spherulites.15 This stage involves the formation of protein aggregates over a wide range of length scales and is characterized by the exponential growth of turbidity and scattered-light intensity.10,13 This has led to the proposal of a double mechanism10,16 involving either secondary nucleation or branching, analogous to that formalized by Eaton, Ferrone, and co-workers to explain sickle cell hemoglobin polymerization17 and recently observed for another hormone, glucagon.18 After incubation for a long time at high temperature, insulin fibrils further reorganize in floccules or large bundles, which eventually precipitate or form a gel at higher concentrations.8-10,14-16,19 In the present work, we study the formation of amyloid fibrillar networks from concentrated solutions of bovine insulin in 20% acetic acid, without added salt, upon incubation at 70 °C. In such (8) Waugh, D. F. J. Cell. Comp. Physiol. 1957, 49, 145–164. (9) Jansen, R.; Dzwolak, W.; Winter, R. Biophys. J. 2005, 88, 1344–1353. (10) Manno, M.; Craparo, E. F.; Podesta, A.; Bulone, D.; Carrotta, R.; Martorana, V.; Tiana, G.; San Biagio, P. L. J. Mol. Biol. 2007, 366, 258–274. (11) Ahmad, A.; Uversky, V. N.; Hong, D.; Fink, A. L. J. Biol. Chem. 2005, 280, 42669–42675. (12) Podesta, A.; Tiana, G.; Milani, P.; Manno, M. Biophys. J. 2006, 90, 589– 597. (13) Manno, M.; Craparo, E. F.; Martorana, V.; Bulone, D.; San Biagio, P. L. Biophys. J. 2006, 90, 4585–4591. (14) Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40, 6036–6046. (15) Krebs, M. R. H.; MacPhee, C. E.; Miller, A. F.; Dunlop, I. E.; Dobson, C. M.; Donald, A. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14420–14424. (16) Librizzi, F.; Rischel, C. Protein Sci. 2005, 14, 3129–3134. (17) Ferrone, F. A.; Hofrichter, J.; Eaton, W. A. J. Mol. Biol. 1985, 183, 611– 631. (18) Andersen, C. B.; Yagi, H.; Manno, M.; Martorana, V.; Ban, T.; Christiansen, G.; Otzen, D. E.; Goto, Y.; Rischel, C. Biophys. J. 2009, 96, 15291536. (19) Pasternack, R. F.; Gibbs, E. J.; Sibley, S.; Woodard, L.; Hutchinson, P.; Genereux, J.; Kristian, K. Biophys. J. 2006, 90, 10331042.
Published on Web 11/13/2009
Langmuir 2010, 26(3), 1424–1426
Manno et al.
Letter
Figure 1. AFM images of a 2 mM insulin solution incubated at 70 °C for 24 h. (Left) 1:102 sample dilution, 1 μm horizontal bar; (right) 1:104 sample dilution, 0.5 μm horizontal bar.
a solvent, insulin is preferentially monomeric and thus the monomer/dimer equilibrium does not complicate the initial stages.11,20 The low ionic strength of the solution increases electrostatic repulsion and thus enhances the formation of elongated objects. It also allows us to obtain experimental kinetics, which are sufficiently slow and reproducible with respect to those observed in insulin solution with salt added.10,14,16 After a few days of incubation at 70 °C, insulin solutions became very viscous because of the formation of large aggregates. A few drops of sample were taken at various dilutions for imaging with atomic force microscopy (AFM). As expected from previous reports,9-16 we observe a network of entangled elongated amyloid fibrils and single (moderately branched) fibrils with a diameter of
