Self-Assembly of Ovalbumin into Amyloid and Non-Amyloid Fibrils Cecile Lara, Simon Gourdin-Bertin, Jozef Adamcik, Sreenath Bolisetty, and Raffaele Mezzenga* Food and Soft Material Science, Institute of Food, Nutrition and Health, ETH Zü rich, Schmelzbergstrasse 9, LFO E 23, 8092 Zü rich, Switzerland * S Supporting Information ABSTRACT: We study the fibrillation pathway of ovalbumin protein and report the simultaneous formation of several types of fibrils, with clear structural and physical differences. We compare the fibrillation mechanisms at low pH with and without salt, and follow the kinetics of fibrils growth by atomic force microscopy (AFM), static and dynamic light scattering (SLS, DLS), and small-angle X-ray scattering (SAXS). We show that, among the morphologies identified, long semiflexible amyloid fibrils (type I), with persistence length L p ∼ 3 μm, Young's modulus E ∼ 2.8 GPa, and cross-β structure are formed. We also observe much more flexible fibrils (type III, L p ∼ 63 nm), that can assemble into multistranded ribbons with time. They show significantly lower intrinsic stiffness (1.1 GPa) and a secondary structure, which is not characteristic of the well-ordered amyloids, as determined by circular dichroism (CD), wide-angle X-ray scattering (WAXS), and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). In between these two main classes of fibrils, a third family, with intermediate flexibility (type II, L p ∼ 300 nm), is also resolved. ■ INTRODUCTION Protein aggregation is one of the most challenging fields in biology and medicine because a large number of human and animal diseases are related to protein self-assembly into amyloid fibrils. 1-3 Non-disease-related amyloids, however, are also of interest in other fields. In food science and technology, for example, protein fibrils offer desirable properties, especially for their interfacial and texture building features. 4-6 In materials science, they provide functional and mechanical properties with applications ranging from medicine to electronics. 7-10 Ovalbumin is one of the most important protein components in egg white and has multifunctional properties, such as its ability to foam and to form gels upon heating. 11,12 The native protein consists of 385 amino acid residues, has a molecular weight of 44500 Da, an isoelectric point of 4.5 and a denaturation temperature of 84 °C at pH 7. 6 Under specific denaturation conditions, such as heat treatment around 75-80 °C, at pH 2 or 7, ovalbumin has been shown to form fibrils in vitro. The fibrils are always reported to have a very flexible morphology with a contour length ranging between 25 and 300 nm, 13-16 depending on the experimental conditions such as pH, temperature, ionic strength, and so on. The fibrillar networks have been shown to undergo gelation, above the critical concentration, for given pH and ionic strength. 17 In some studies, a decrease of α-helical content, binding of thioflavin T to the ovalbumin aggregates, 17 or of Congo red, 18 suggest the formation of β-sheet type of structures. In this work, however, we combine single molecule microscopy technique with bulk scattering and spectroscopic techniques, to demonstrate that, at pH 2 and 90 °C, ovalbumin does not only form the very flexible fibrils morphology reported so far (here referred as type III), but also a longer and stiffer type of fibrils is formed. Only this latter class of fibrils exhibits the typical fingerprint of amyloid fibrils, in direct analogy with amyloid fibrils assembled from other globular food proteins such as dialyzed β-lactoglobulin or lysozyme. 19-21 This class of micrometers-long semiflexible and rigid fibrils, with high β- sheet content, is identified for the first time from the ovalbumin protein, and referred in what follows as type I fibrils. Among the various types of fibrillar aggregates, a third main morphology (type II), with intermediate thickness and flexibility, is also resolved. The fibrillation process depends on several parameters, such as the pH, which affects the protein net charge and hydrolysis upon heat treatment, 22 and the ionic strength of the solution, with counterions influencing the proteins electrostatic inter- actions. 13 We therefore compare the kinetics of fibrils formation in two conditions: with and without addition of 50 mM NaCl. The distribution of the fibrils contour lengths and their persistence lengths, at the two conditions of low and high ionic strength, as well as their Young's modulus distribution were determined by AFM images analysis and AFM peak force nanoindentation, respectively. Scattering techniques such as small-angle X-ray scattering (SAXS), wide-angle X-ray scatter- ing (WAXS), and dynamic and static light scattering (DLS, SLS) were used to characterize further the structural features in solution or in the dry state. Mass spectrometry (MALDI MS) and gel electrophoresis (SDS-PAGE) were also used to get a better understanding of the ovalbumin self-assembly process at the peptide sequences length scale. Finally, Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) gave evidence of the secondary structure differences between the two extreme types of fibrils: type I (semiflexible) amyloids and type III (worm-like) flexible fibrils. Received: September 20, 2012 Revised: October 24, 2012 Published: October 25, 2012 Article pubs.acs.org/Biomac © 2012 American Chemical Society 4213 dx.doi.org/10.1021/bm301481v | Biomacromolecules 2012, 13, 4213-4221