The aggregation of proteins is central to many aspects of daily life, including food processing, blood coagulation, eye cataract formation disease and prion-related neurodegenerative infections [1][2][3][4][5] . However, the physical mechanisms responsible for amyloidosis-the irreversible fibril formation of various proteins that is linked to disorders such as Alzheimer's, Creutzfeldt-Jakob and Huntington's diseases-have not yet been fully elucidated [6][7][8][9] . Here, we show that different stages of amyloid aggregation can be examined by performing a statistical polymer physics analysis of single-molecule atomic force microscopy images of heat-denatured b-lactoglobulin fibrils. The atomic force microscopy analysis, supported by theoretical arguments, reveals that the fibrils have a multistranded helical shape with twisted ribbon-like structures. Our results also indicate a possible general model for amyloid fibril assembly and illustrate the potential of this approach for investigating fibrillar systems.Protein self-assembly is a wide-ranging phenomenon and is of great importance in several areas of science. The reversible formation of fibrils from globular proteins is a phenomenon occurring naturally, in vivo, for proteins such as actin and tubulin 10,11 . Other well-known examples of protein fibrillation include the irreversible amyloid fibril formation of various proteins implicated in neurological disorders such as Alzheimer's, Creutzfeldt-Jakob or Huntington's diseases. Typically, these fibrils have long, unbranched, and often twisted structures that are a few nanometres in diameter 1,8 . However, many peptides and proteins, including many globular food proteins that are used as gelling agents, foaming agents or emulsifiers, can also form amyloid-like structures in vitro. Such structures can possess desirable mechanical properties and can be used to create useful textures and structures 12,13 .An example of a fibril formation of globular proteins is provided by the fine-stranded heat-set gels formed by heating solutions of various globular food proteins such as ovalbumin, bovine serum albumin 12 and b-lactoglobulin 13 . b-Lactoglobulin has been particularly well studied, because it represents both a relevant model system and a major whey protein of interest to the food industry [14][15][16][17][18][19][20][21][22] .Despite the fact that the individual parameters controlling the aggregation of globular proteins into amyloid fibrils have been well identified, the driving force for such an aggregation process still remains obscure. Studies devoted to the characterization of fibril structures based on light, neutrons and X-ray scattering methods, being bulk techniques, have only provided an average ensemble picture of the fibrils 23 . Single-molecule techniques such as atomic force microscopy (AFM) and transmission electron microscopy (TEM) have recently emerged to probe amyloid fibrils at the molecular level [24][25][26][27] . Nevertheless, so far, a fully comprehensive picture of the aggregation behaviour...

We report on the conformation of heat-induced bovine beta-lactoglobulin (betalg) aggregates prepared at different pH conditions, and their complexes with model anionic surfactants such as sodium dodecyl sulfate (SDS). The investigation was carried out by combining a wide range of techniques such as ultra small angle light scattering, static and dynamic light scattering, small angle neutron scattering, small-angle X-ray scattering, electrophoretic mobility, isothermal titration calorimetry (ITC) and transmission electron microscopy. Three types of aggregates were generated upon heating betalg aqueous dispersions at increasing pH from 2.0 to 5.8 to 7.0: rod-like aggregates, spherical aggregates, and worm-like primary aggregates, respectively. These aggregates were shown not only to differ for their sizes and morphologies, but also for their internal structures and fractal dimensions. The main differences between aggregates are discussed in terms of the ionic charge and conformational changes arising for betalg at different pHs. The formation of complexes between SDS and the various protein aggregates at pH 3.0 was shown to occur by two main mechanisms: at low concentration of SDS, the complex formation occurs essentially by ionic binding between the positive residues of the protein and the negative sulfate heads of the surfactant. At complete neutralization of charges, precipitation of the complexes is observed. Upon further increase in SDS concentration, complex formation of SDS and the protein aggregates occurs primarily by hydrophobic interactions, leading to (i) the formation of an SDS double layer around the protein aggregates, (ii) the inversion of the total ionic charge of each individual protein aggregate, and (iii) the complete redispersion of the protein aggregate-SDS complexes in water. Remarkably, the SDS double layer around the protein aggregates provides an efficient protective shield, preventing precipitation of the aggregates at any possible pH values, including those values corresponding to the isoelectric pH of the aggregates.

We have developed a new method allowing the study of the thermodynamic phase behavior of mesoscopic colloidal systems consisting of amyloid protein fibers in water, obtained by heat denaturation and aggregation of β-lactoglobulin, a dairy protein. The fibers have a cross section of about 5.2 nm and two groups of polydisperse contour lengths: (i) long fibers of 1-20 μm, showing semiflexible behavior, and (ii) short rods of 100-200 nm long, obtained by cutting the long fibers via high-pressure homogenization. At pH 2 without salt, these fibers are highly charged and stable in water. We have studied the isotropic-nematic phase transition for both systems and compared our results with the theoretical values predicted by Onsager's theory. The experimentally measured isotropic-nematic phase transition was found to occur at 0.4% and at 3% for the long and short fibers, respectively. For both systems, this phase transition occurs at concentrations more than 1 order of magnitude lower than what is expected based on Onsager's theory. Moreover, at low enough pH, no intermediate biphasic region was observed between the isotropic phase and the nematic phase. The phase diagrams of both systems (pH vs concentration) showed similar, yet complex and rich, phase behavior. We discuss the possible physical fundamentals ruling the phase diagram as well as the discrepancy we observe for the isotropic-nematic phase transition between our experimental results and the predicted theoretical results. Our work highlights that systems formed by water-amyloid protein fibers are way too complex to be understood based solely on Onsager's theories. Experimental results are revisited in terms of the Flory's theory (1956) for suspensions of rods, which allows accounting for rod-solvent hydrophobic interactions. This theoretical approach allows explaining, on a semiquantitative basis, most of the discrepancies observed between the experimental results and Onsager's predictions. The sources of protein fibers complex colloidal behavior are analyzed and discussed at length.

Interfacial properties of native β-lactoglobulin monomers and their heat-induced fibers, of two different lengths, were investigated at pH 2, through surface tension measurements at water-air and water-oil interfaces and interfacial shear rheology at the water-oil interface. The applied heat treatment generates a mixed system of fibers with unconverted monomers and hydrolyzed peptides. The surface tension of this system at the water-air interface decreased more rapidly than the surface tension of native monomers, especially at short times (10(-3) to 10(2) s). This behavior was not observed when the unconverted monomers and peptides were removed by dialysis. At the water-oil interface, the adsorption kinetics was much faster than at the water-air interface, with a plateau interfacial pressure value reached after 1 h of adsorption. For all the systems, interfacial shear rheology showed the formation of a highly elastic interface, with solid-like behavior at 1-10(3) s time scales. The highest modulus was observed for the long fibers and the lowest for the native monomers. Creep-compliance curves in the linear regime could be reduced to a single master curve, showing similar spectra of relaxation times for all investigated systems. Upon large deformations, the interfaces formed with long fibers showed the most rigid and fragile behavior. This rigidity was even more pronounced in the presence of unconverted monomers.

We have investigated the thermodynamic and dynamic behavior of multistranded β-lactoglobulin protein fibrils in water, by combining static, dynamic, and depolarized dynamic light scattering (SLS, DLS, DDLS), small angle neutron scattering (SANS), rheology, and cryogenic transmission electron microscopy (cryo-TEM). We focus on the region of the phase diagram at which ionic strength and concentration changes induce transitions in gelation and lyotropic liquid crystalline behavior. An increase in ionic strength, induced by NaCl salt, progressively causes the phase transitions from nematic (N) to gel (G) phases; a further increase causes the transition to a translucent phase and to a macroscopic phase separation, respectively. An increase in fibril concentration induces first a phase transition from an isotropic (I) to a nematic phase (N); a further increase induces the formation of a gel phase. The protein gel strength is investigated by rheology measurements. SANS and osmotic compressibility calculated by SLS measurements clearly capture the main features of the IN transition of β-lactoglobulin protein fibrils. The form and structure factors measured by scattering experiments are analyzed by the polymer reference interaction site model (PRISM). Dynamics of the protein fibrils at different concentrations, measured by polarized and depolarized dynamic light scattering, show both individual and collective diffusion after the isotropic-nematic transition. Above this transition, cryo-TEM images further demonstrate the alignment of the protein fibrils, which is quantified by a 2D order parameter. This work discusses comprehensively, both experimentally and theoretically, the thermodynamics and dynamic features of β-lactoglobulin amyloid fibrils in a vast region of the concentration-ionic strength phase diagram.

We investigate the effects of variable linear charge density and Debye length on the mesoscopic properties of beta-lactoglobulin fibers in water, by changing the pH and ionic strength, respectively. We determine the isotropic-nematic (I-N) transition by cross-polarized microscopy and quantify by atomic force microscopy the increasing tendency of the fibers to aggregate upon raising ionic strength. We then compare experimental I-N transitions with theoretical expected values based on Onsager theory. Unlike previous reports on lyotropic liquid crystalline behavior of protein fibers, we show that, if double layer effects and aggregation of fibers are correctly included directly in the second virial coefficient and excluded volume, Onsager theory accurately predicts the experimental I-N transition versus pH and ionic strength.

In this paper, we examined the characteristic behavior of localized surface plasmon resonances (LSPR) of Au dot and ring arrays in response to the selective binding of biomolecules. To do this, patterned arrays of Au rings and dots with various feature scales were fabricated over large areas by an imprint lithography technique. Our results showed that the LSPR spectra of the Au nanorings exhibited a blue shift with increase in the ring widths and asymptotically converged to those for Au nanodots. This clearly implies that the LSPR spectra can be tuned over an extended wavelength range by varying the ring width. For an illustrative purpose, the patterned Au structures were used to detect the binding of streptavidin to biotin. In doing this, the Au patterns were chemically modified with G4 dendrimers of amine terminated poly(amidoamine), which facilitated the tethering of biotin onto the Au pattern. Exposure of the biotinylated Au nanorings to aqueous streptavidin solution induced both red-shifts of the LSPR spectra and changes in the peak intensities. The sensitivity of the LSPR spectra to the binding of the biomolecules was enhanced as the ring width of Au rings was decreased.

Nanotechnology as well as advanced microscopy can play a fundamental role in understanding biological mechanisms. Here we present a study that combines a new type of nanomaterial with a new type of microscopy and highlights the potential for gathering novel information about cell membrane penetration and cytosol local viscosity. On the material side, we used gold nanoparticles that have an ordered stripe-like arrangement of domains. These "striped" nanoparticles are able to penetrate cell membranes directly without porating them. On the microscopy side, we used photothermal heterodyne imaging which allows detection of individual nanometer-sized gold particles in complex media. We showed that we can probe cytosolic presence as well as dynamics of these nanoparticles even at very low concentrations. We used the fluctuations of the photothermal signal from particles diffusing in the detection volume to estimate local cytosol viscosity which is about 20 times larger than that of water. This work opens new perspectives for mapping local diffusion properties of nano-objects inside living cells.