Surface elastic properties of human retinal pigment epithelium melanosomes

Atomic force microscope (AFM) imaging and nanoindentation measurements in water were used to probe the mechanical properties of retinal pigment epithelium melanosomes isolated from 14-year-old and 76-year-old donors. Topographic imaging reveals surface roughness similar to previous measurements on dry melanosomes. Force-indentation measurements show different types of responses that were catalogued into four different categories. In these measurements no permanent surface damage of melanosomes was observed as revealed by imaging before and after indentation measurements. The indentation measurements that exhibited nearly elastic responses were used to determine the Young's modulus of melanosomes. The average Young's modulus values are similar for 14-year-old and 76-year-old melanosomes with a somewhat narrower distribution for the 14-year-old sample. These elastic modulus values are considerably higher than the modulus of organelles with cytoplasm (<1 MPa) and approaching values of the modulus of protein crystals (approximately 100 MPa) indicating rather high packing density of biologic material in melanosomes. The width of the Young's modulus distributions is considerable spanning from few megapascals to few tens of megapascals indicating large heterogeneity in the structure. A fraction of the force curves cannot be described by the homogeneous elastic sample model; these force curves are consistent with approximately 10 nm structural heterogeneity in melanosomes. The approach-withdraw hysteresis indicates a significant viscoelasticity, particularly in the samples from the 14-year-old sample. Adhesion of the AFM probe was detected on approximately 3% and approximately 20% of the surface of 14-year-old and 76-year-old samples, respectively. In light of previous studies on these same melanosomes using photoelectron emission microscopy, this adhesion is attributed to the presence of lipofuscin on the surface of the melanosomes. This suggestion indicates that part of the difference in photochemical properties between the old and young melanosomes originates from surface lipofuscin.     

Micromechanical testing of individual collagen fibrils

A novel method based on AFM was used to attach individual collagen fibrils between a glass surface and the AFM tip, to allow force spectroscopy studies of these. The fibrils were deposited on glass substrates that are partly coated with Teflon AF. A modified AFM tip was used to accurately deposit epoxy glue droplets on either end of the collagen fibril that cross the glass-Teflon AF interface, as to such attach it with one end to the glass and the other end to the AFM tip. Single collagen fibrils have been mechanically tested in ambient conditions and were found to behave reversibly up to stresses of 90 MPa. Within this regime a Young's modulus of 2-7 GPa was obtained. In aqueous media, the collagen fibrils could be tested reversibly up to about 15 MPa, revealing Young's moduli ranging from 0.2 to at most 0.8 GPa.     

Nanoscale interfibrillar adhesion in UHMWPE fibers

The goal of this research is to quantify the fibrillar adhesive energy in ultra‐high molecular weight polyethylene fibers, characteristic of nanoscale fibril interactions. Quantification of these energies is vital to the understanding of fibrillar deformation mechanisms that have been shown to play an important role in fiber performance. This is achieved through the development and implementation of a nanosplitting technique developed through the use of AFM‐enabled nanoindentation. This technique allows the quantification of nanoscale adhesive energies through careful monitoring of load and unload curves as well as examination of the residual split through high‐resolution AFM images. Results indicate that the average nanoscale fibril adhesive energy is over 3 times larger than the energy expected from van der Waals interactions alone. This indicates that a significant degree of physical interactions exist between fibrils, beyond van der Waals interactions, in the form of tie‐molecules, fibrillar network junctions, and bridging lamellar crystals. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 391–401     

Absolute structural constraints on amyloid fibrils from solid-state NMR spectroscopy of partially oriented samples

We demonstrate that absolute, molecular-level structural information can be obtained from solid-state NMR measurements on partially oriented amyloid fibrils. Specifically, we show that the direction of the fibril axis relative to a carbonyl 13C chemical shift anisotropy (CSA) tensor can be determined from magic-angle spinning (MAS) sideband patterns in 13C NMR spectra of fibrils deposited on planar substrates. Deposition of fibrils on a planar substrate creates a highly anisotropic distribution of fibril orientations (hence, CSA tensor orientations) with most fibrils lying in the substrate plane. The anisotropic orientational distribution gives rise to distorted spinning sideband patterns in MAS spectra from which the fibril axis direction can be inferred. The experimentally determined fibril axis direction relative to the carbonyl CSA tensor of Val12 in fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta1-40) agrees well with the predictions of a recent structural model (Petkova et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16742-16747) in which Val12 is contained in a parallel beta-sheet in the cross-beta motif characteristic of amyloid fibrils.     

AFM nanoindentation of viscoelastic materials with large end‐radius probes

We used atomic force microscopy (AFM) nanoindentation to measure mechanical properties of polymers. Although AFM is generally acknowledged as a high‐resolution imaging tool, accurate quantification of AFM nanoindentation results is challenging. Two main challenges are determination of the projected area for objects as small as AFM tips and use of appropriate analysis methods for viscoelastic materials. We report significant accuracy improvements for modulus measurements when large end‐radius tips with appropriate cantilever stiffnesses are used for indentation. Using this approach, the instantaneous elastic modulus of four polymers we studied was measured within 30 to 40% of Dynamic Mechanical Analysis (DMA) results. The probes can, despite their size and very high stiffnesses, be used for imaging of very small domains in heterogeneous materials. For viscoelastic materials, we developed an AFM creep test to determine the instantaneous elastic modulus. The AFM method allows application of a nearly perfect stepload that facilitates data analysis based on hereditary integrals. Results for three polymers suggest that the observed creep in the materials has a strong plastic flow component even at small loads. In this respect, the spherical indenter tips behave like “sharp” indenters used in indentation studies with instrumented indenters. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 1573–1587, 2009     

AFM nanoindentation to determine Young’s modulus for different EPDM elastomers

AFM nanoindentation was investigated as a method for determining the micromechanical properties of polymer materials. It is generally accepted that the shape of the tip of the cantilever undergoes a change in a standard AFM setup. The shape defines the projected contact area, so it is a parameter directly proportional to the elastic modulus; any change in the shape thus affects the accuracy of the results. The method suggested in this paper relies on the introduction of an experimentally determined tip-area function. Values for Young’s modulus were calculated for EPDM samples with different degrees of cure and crystallinity. The degree of crystallinity has a greater impact on the mechanical properties of the material than the degree of cure. Depending on the amplitude of the indentation, the E-moduli determined by AFM are systematically higher. When studying different regions of polymer materials, the values of the E-modulus determined by AFM become identical to those measured by means of DMA on extrapolation of the modulus at zero indentation.     

Nanoindentation as a Probe for Mechanically‐Induced Molecular Migration in Layered Organic Donor–Acceptor Complexes

Nanoindentation and scratch experiments on 1:1 donor–acceptor complexes, 1 and 2 , of 1,2,4,5‐tetracyanobenzene with pyrene and phenanthrene, respectively, reveal long‐range molecular layer gliding and large interaction anisotropy. Due to the layered arrangements in these crystals, these experiments that apply stress in particular directions result in the breaking of interlayer interactions, thus allowing molecular sheets to glide over one another with ease. Complex 1 has a layered crystal packing wherein the layers are 68° skew under the (002) face and the interlayer space is stabilized by van der Waals interactions. Upon indenting this surface with a Berkovich tip, pile‐up of material was observed on just one side of the indenter due to the close angular alignment of the layers with the half angle of the indenter tip (65.35°). The interfacial differences in the elastic modulus (21 %) and hardness (16 %) demonstrate the anisotropic nature of crystal packing. In 2 , the molecular stacks are arranged in a staggered manner; there is no layer arrangement, and the interlayer stabilization involves C? H???N hydrogen bonds and π???π interactions. This results in a higher modulus (20 %) for (020) as compared to (001), although the anisotropy in hardness is minimal (4 %). The anisotropy within a face was analyzed using AFM image scans and the coefficient of friction of four orthogonal nanoscratches on the cleavage planes of 1 and 2 . A higher friction coefficient was obtained for 2 as compared to 1 even in the cleavage direction due to the presence of hydrogen bonds in the interlayer region making the tip movement more hindered.     

Structure and mechanical properties of poly(L‐lactic acid) crystals and fibers

The elastic constants of poly(L ‐lactic acid) (PLLA) crystals are reported on the basis of a commercial software package and the published crystal structure of the α form. A chain modulus of 36 GPa and a shear modulus of 3 GPa have been obtained for cylindrically symmetric aggregates of perfectly oriented crystals. The helical conformation of the PLLA molecule reduces the stiffness in the chain axis direction because bond rotation plays a significant role in the deformation. X‐ray crystal strain measurements suggest that shear of the α crystal parallel to the helix axis is the easiest mode of deformation, in agreement with the expectations obtained from the low shear modulus of 3 GPa obtained from the theoretical calculations. A combination of small‐ and wide‐angle X‐ray scattering, differential scanning calorimetry, dynamic mechanical thermal analysis, and shrinkage measurements has been used to characterize the structure that develops and the crystal transformation that occurs during fiber processing. The structure that develops during processing very much depends on the crystal transformation, and a structural model is proposed for fibers at different degrees of plastic deformation. The transformation of the α crystal into the β form and vice versa is governed primarily by shear along the helix axis because the chains must shear past each other during the crystal transformation, disrupting the lamellar packing. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 45: 892–902, 2007     

Monitoring and inhibition of insulin fibrillation by a small organic fluorogen with aggregation-induced emission characteristics

Amyloid fibrillation of proteins is associated with a great variety of pathologic conditions. Development of new molecules that can monitor amyloidosis kinetics and inhibit fibril formation is of great diagnostic and therapeutic value. In this work, we have developed a biocompatible molecule that functions as an ex situ monitor and an in situ inhibitor for protein fibrillation, using insulin as a model protein. 1,2-Bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene salt (BSPOTPE) is nonemissive when it is dissolved with native insulin in an incubation buffer but starts to fluoresce when it is mixed with preformed insulin fibril, enabling ex situ monitoring of amyloidogenesis kinetics and high-contrast fluorescence imaging of protein fibrils. Premixing BSPOTPE with insulin, on the other hand, inhibits the nucleation process and impedes the protofibril formation. Increasing the dose of BSPOTPE boosts its inhibitory potency. Theoretical modeling using molecular dynamics simulations and docking reveals that BSPOTPE is prone to binding to partially unfolded insulin through hydrophobic interaction of the phenyl rings of BSPOTPE with the exposed hydrophobic residues of insulin. Such binding is assumed to have stabilized the partially unfolded insulin and obstructed the formation of the critical oligomeric species in the protein fibrillogenesis process.     

Nanoindentation in Crystal Engineering: Quantifying Mechanical Properties of Molecular Crystals

Nanoindentation is a technique for measuring the elastic modulus and hardness of small amounts of materials. This method, which has been used extensively for characterizing metallic and inorganic solids, is now being applied to organic and metal–organic crystals, and has also become relevant to the subject of crystal engineering, which is concerned with the design of molecular solids with desired properties and functions. Through nanoindentation it is possible to correlate molecular‐level properties such as crystal packing, interaction characteristics, and the inherent anisotropy with micro/macroscopic events such as desolvation, domain coexistence, layer migration, polymorphism, and solid‐state reactivity. Recent developments and exciting opportunities in this area are highlighted in this Minireview.     

Polymorphism of Amyloid Fibrils Induced by Catalytic Seeding: A Vibrational Circular Dichroism Study

Amyloidal protein fibrils occur in many biological events, but their formation and structural variability are understood rather poorly. We systematically explore fibril polymorphism for polyglutamic acid (PGA), insulin and hen egg white lysozyme. The fibrils were grown in the presence of “seeds”, that is fibrils of the same or different protein. The seeds in concentrations higher than about 5 % of the total protein amount fully determined the structure of the final fibrils. Fibril structure was monitored by vibrational circular dichroism (VCD) spectroscopy and other techniques. The VCD shapes significantly differ for different fibril samples. Infrared (IR) and VCD spectra of PGA were also simulated using density functional theory (DFT) and a periodic model. The simulation provides excellent basis for data interpretation and reveals that the spectral shapes and signs depend both on fibril length and twist. The understanding of fibril formation and interactions may facilitate medical treatment of protein misfolding diseases in the future.     

On collagen II fibrillogenesis

Collagen fibrils exhibit noticeable differences in their molecular order in the direction perpendicular to fibril axis. For instance, three-dimensional crystallinity prevails for type I collagen (e.g. tendons) whereas only liquid-like order in the lateral direction prevails for type II collagen (e.g. cartilage). The latter situation has been likened to that of a smectic liquid crystal. It is suggested here that the fibrillogenesis of type II collagen is indeed directed by a possibly metastable liquid crystalline mesophase involving a supramolecular assembling process. The occurrence of decoration on the fibrillar surface enhances a liquid crystalline phase due to preferential growth assumed to continue along the axial direction, and an increasing persistence length with respect to triple-helical collagen molecules.     

Variably elastic hydrogel patterned via capillary action in microchannels

Agarose hydrogels of varied elastic modulus can be patterned into 100-microm-wide channels with wall heights of 60 microm. After modifying the hydrogels with chloroacetic acid (acid gels), they are amenable to modification with amine-containing ligands using EDC-NHS chemistry. Using both rheometry and atomic force microscopy (AFM) nanoindentation measurements, the elastic modulus of unmodified hydrogels increases linearly from 3.6 +/- 0.5 kPa to 45.2 +/- 5.5 kPa for 0.5 to 2.0 wt/vol % hydrogel, respectively. The elastic modulus of acid gels is 2.2 +/- 0.3 kPa to 16.2 +/- 1.6 kPa for 0.5 to 2.0 wt/vol %, respectively. No further changes were measured after further modifying the acid gels with fibronectin. Confocal images of rhodamine-modified acid gels show that the optimal filling viscosity of the agarose solutions is between 1 and 4 cP. This new method of patterning allows for the creation of substrates that take advantage of both micron-scale patterns and variably elastic hydrogels.     

The unique Alzheimer's β-amyloid triangular fibril has a cavity along the fibril axis under physiological conditions

Elucidating the structure of Aβ(1-40) fibrils is of interest in Alzheimer's disease research because it is required for designing therapeutics that target Aβ(1-40) fibril formation at an early stage of the disease. M35 is a crucial residue because of its potential oxidation and its strong interactions across β-strands and across β-sheets in Aβ fibrils. Experimentally, data for the three-fold symmetry structure of the Aβ(9-40) fibril suggest formation of tight hydrophobic core through M35 interactions across the fibril axis and strong I31-V39 interactions between different cross-β units. Herein, on the basis of experimental data, we probe conformers with three-fold symmetry of the full-length Aβ(1-40). Our all-atom molecular dynamics simulations in explicit solvent of conformers based on the ssNMR data reproduced experimental observations of M35-M35 and I31-V39 distances. Our interpretation of the experimental data suggests that the observed ～5-7 ? M35-M35 distance in the fibril three-fold symmetry structure is likely to relate to M35 interactions along the fibril axis, rather than across the fibril axis, since our measured M35-M35 distances across the fibril axis are consistently above 15 ?. Consequently, we revealed that the unique Aβ(1-40) triangular structure has a large cavity along the fibril axis and that the N-termini can assist in the stabilization of the fibril by interacting with the U-turn domains or with the C-termini domains. Our findings, together with the recent cyroEM characterization of the hollow core in Aβ(1-42) fibrils, point to the relevance of a cavity in Aβ(1-40/1-42) oligomers which should be considered when targeting oligomer toxicity.     

Influence of drying restraint on physical and mechanical properties of nanofibrillated cellulose films

Nanofibrillated cellulose (NFC) is a renewable and biodegradable fibril that possesses high strength and stiffness resulting from high level hydrogen bonding. Films made from NFC shrink and distort as they transition from a wet state (20 wt% solids) to a state of moisture equilibrium (90 wt% solids at 50 % RH, 23 °C). Material distortions are driven by development of moisture gradients within the fibril network and effectively reduce mechanical performance. For this study, NFC was extracted from softwood holocellulose by first employing a chemical pretreatment [(2,2,6,6-tetramethylpiperidin-1-yl)oxyl catalyzed oxidation] followed by mechanical fibrillation using ultrasound energy. To assess the problem of film distortion, neat NFC films were dried at 50 % RH, 23 °C under one of the following three restraint conditions: fully restrained, partially restrained, and uniaxially drawn. The influence of restraint condition on the resulting physical and mechanical properties was evaluated. Raman and X-ray results showed that fibrils in the uniaxially drawn specimens tended to align with the drawing axis, whereas no in-plane orientation effects were observed for the fully or partially restrained specimens. Fully restrained specimens had a respective strength and stiffness of 222 MPa and 14 GPa in every (in-plane) direction. However, samples that were wet-drawn to a 30 % strain level had a respective strength and stiffness of 474 MPa and 46 GPa in the direction of draw. Mechanical properties for axially drawn specimens had both fibril alignment and fibril straightening contributions.     

On collagen II fibrillogenesis

Collagen fibrils exhibit noticeable differences in their molecular order in the direction perpendicular to fibril axis. For instance, three‐dimensional crystallinity prevails for type I collagen (e.g. tendons) whereas only liquid‐like order in the lateral direction prevails for type II collagen (e.g. cartilage). The latter situation has been likened to that of a smectic liquid crystal. It is suggested here that the fibrillogenesis of type II collagen is indeed directed by a possibly metastable liquid crystalline mesophase involving a supramolecular assembling process. The occurrence of decoration on the fibrillar surface enhances a liquid crystalline phase due to preferential growth assumed to continue along the axial direction, and an increasing persistence length with respect to triple‐helical collagen molecules.     

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid β (1–40)

A small library of rationally designed amyloid β [Aβ(1–40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aβ(1–40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid‐state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aβ(1–40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aβ fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross‐β structure.     

常压动态保压注塑自增强高密度聚乙烯的结构与性能

研究了常压动态保压注塑自增强高密度聚乙烯的结构和力学性能的关系,自增强高密度聚乙烯的抗张强度和杨氏模量分别从原来的23MPa和1.0GPa提高到93MPa和5.0GPa。DSC、TEM和X射线衍射研究结果表明:力学性能的显著改善主要归因于串晶的产生、高分子链沿流动方向的轴取向和结构更加完善的球晶的生成。和高压保压法相比,本法具有现实的工业应用前景。     

Structure and Composition of Insulin Fibril Surfaces Probed by TERS

Amyloid fibrils associated with many neurodegenerative diseases are the most intriguing targets of modern structural biology. Significant knowledge has been accumulated about the morphology and fibril-core structure recently. However, no conventional methods could probe the fibril surface despite its significant role in the biological activity. Tip-enhanced Raman spectroscopy (TERS) offers a unique opportunity to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range. Herein, TERS is utilized for characterizing the secondary structure and amino acid residue composition of the surface of insulin fibrils. It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations. More than 30% of the fibril surface is dominated by β-sheet secondary structure, further developing Dobson's model of amyloid fibrils (Jimenez et al. Proc. Natl. Acad. Sci. U.S.A. 2002 , 99 , 9196 - 9201 ). The propensity of various amino acids to be on the fibril surface and specific surface secondary structure elements were evaluated. β-sheet areas are rich in cysteine and aromatic amino acids, such as phenylalanine and tyrosine, whereas proline was found only in α-helical and unordered protein clusters. In addition, we showed that carboxyl, amino, and imino groups are nearly equally distributed over β-sheet and α-helix/unordered regions. Overall, this study provides valuable new information about the structure and composition of the insulin fibril surface and demonstrates the power of TERS for fibril characterization.     

Aβ40 and Aβ42 amyloid fibrils exhibit distinct molecular recycling properties

A critical aspect to understanding the molecular basis of Alzheimer's disease (AD) is the characterization of the kinetics of interconversion between the different species present during amyloid-β protein (Aβ) aggregation. By monitoring hydrogen/deuterium exchange in Aβ fibrils using electrospray ionization mass spectrometry, we demonstrate that the Aβ molecules comprising the fibril continuously dissociate and reassociate, resulting in molecular recycling within the fibril population. Investigations on Aβ40 and Aβ42 amyloid fibrils reveal that molecules making up Aβ40 fibrils recycle to a much greater extent than those of Aβ42. By examining factors that could influence molecular recycling and by running simulations, we show that the rate constant for dissociation of molecules from the fibril (k(off)) is much greater for Aβ40 than that for Aβ42. Importantly, the k(off) values obtained for Aβ40 and Aβ42 reveal that recycling occurs on biologically relevant time scales. These results have implications for understanding the role of Aβ fibrils in neurotoxicity and for designing therapeutic strategies against AD.