The formation of amyloid fibrils from proteins in the lysozyme family

Amyloid fibrils are highly ordered protein assemblies known to contribute to the pathology of a variety of genetic and aging-associated diseases. More recently, these fibrils have been shown to be useful as structural scaffolds in both natural biological systems and nanotechnology applications. The intense interest in amyloid fibrils has led to the investigation of well-characterized proteins, such as hen egg white lysozyme (HEWL), as model systems to examine structural and mechanistic principles that may be generally applicable to all amyloid fibrils. The purpose of this review is to critically examine the fibril-formation literature of proteins in the lysozyme family with respect to the known structure and folding properties of these proteins. The goal is to identify similarities and differences within the family, examine general misfolding / aggregation principles, and identify key areas of importance for future work on the fibril formation of these proteins.     

Elucidation of Metal‐Ion Accumulation Induced by Hydrogen Bonds on Protein Surfaces by Using Porous Lysozyme Crystals Containing RhIII Ions as the Model Surfaces

Metal‐ion accumulation on protein surfaces is a crucial step in the initiation of small‐metal clusters and the formation of inorganic materials in nature. This event is expected to control the nucleation, growth, and position of the materials. There remain many unknowns, as to how proteins affect the initial process at the atomic level, although multistep assembly processes of the materials formation by both native and model systems have been clarified at the macroscopic level. Herein the cooperative effects of amino acids and hydrogen bonds promoting metal accumulation reactions are clarified by using porous hen egg white lysozyme (HEWL) crystals containing Rh^III ions, as model protein surfaces for the reactions. The experimental results reveal noteworthy implications for initiation of metal accumulation, which involve highly cooperative dynamics of amino acids and hydrogen bonds: i) Disruption of hydrogen bonds can induce conformational changes of amino‐acid residues to capture Rh^III ions. ii) Water molecules pre‐organized by hydrogen bonds can stabilize Rh^III coordination as aqua ligands. iii) Water molecules participating in hydrogen bonds with amino‐acid residues can be replaced by Rh^III ions to form polynuclear structures with the residues. iv) Rh^III aqua complexes are retained on amino‐acid residues through stabilizing hydrogen bonds even at low pH (≈2). These metal–protein interactions including hydrogen bonds may promote native metal accumulation reactions and also may be useful in the preparation of new inorganic materials that incorporate proteins.     

L-谷氨酸与维多利亚绿S作用机理及应用研究

用紫外-可见光度法、共振散射光谱法探讨了在弱酸性介质中,碱性染料维多利亚绿S与酸性氨基酸L-谷氨酸相互作用机理,认为L-谷氨酸分子通过盐键和氢键相互结合成含有亲水性基团的分子链,这条分子链可以将维多利亚绿S的疏水性基团包在里面,使其亲水性基团朝外,并通过盐键和氢键与维多利亚绿S作用形成大的聚合体,其聚合体体积远远大于染料自身聚集体的体积,使溶液颜色变深,分子光谱吸收值增加.并优化了反应体系酸度、反应物用量、反应时间、加试剂顺序等影响反应速度的因素,确定了最佳实验条件,建立了测定L-谷氨酸的方法.线性范围为10mg/L～100 mg/L,相关系数为0.9989.直接测定样品中谷氨酸获得满意结果.     

Supramolecular Regulation of Polydopamine Formation by Amyloid Fibers

Polydopamine (PD) and melanin species are chemically complex systems, the formation and properties of which are incompletely understood. Inspired by the role of functional amyloids in melanin biosynthesis, this paper examines the influences of the supramolecular structure of amyloids on oxidative polymerization of dopamine. Kinetic analyses on the formation of PD species in the presence of hen egg white lysozyme (HEWL) fibers or soluble HEWL revealed that both forms gave rise to the total quantity of PD species, but the rate of their formation could be accelerated only by the amyloid form. PD species formed with HEWL fibers showed a morphology of bundled fibers, whereas those with soluble HEWL had a mesh-like structure. Amyloid fibers of recombinant Pmel17 had properties similar to those of HEWL fibers in modulating PD formation. The results presented here suggest how nature designs functionality with an amyloid structure and can help understand and engineer chemistries of other functional amyloids.     

Dimers of formic acid, acetic acid, formamide and pyrrole-2-carboxylic acid: an ab initio study

The intermolecular hydrogen bonds in dimers of formic acid, acetic acid, and formamide were investigated. Additionally, three configurations of the pyrrole-2-carboxylic acid (PCA) dimer were studied to analyze how the pyrrole pi-electron system influences the carboxylic groups connected by double O-H...O hydrogen bonds. The ab initio calculations for the systems investigated were performed at MP2/6-311++G(d,p), MP2/aug-cc-pVDZ, and MP2/aug-cc-pVTZ//MP2/aug-cc-pVDZ levels of theory. The "atoms in molecules" theory of Bader was used and the analysis of the critical points was performed to study the nature of hydrogen bonds. The decomposition of the total interaction energy applied here reveals that the delocalization energy term is a particularly important attractive contribution in these systems, more important in the case of systems forming homonuclear O-H...O double hydrogen bonds than in the case of those connected through heteronuclear N-H...O bonds. Because the systems analyzed may be formally classified as the resonance-assisted hydrogen bonds (RAHBs), it seems that the dominant contribution from the delocalization interaction energy term is a distinguished feature of such interactions.     

Amyloid‐β Peptide Induces Prion Protein Amyloid Formation: Evidence for Its Widespread Amyloidogenic Effect

Transmissible spongiform encephalopathy is associated with misfolding of prion protein (PrP) into an amyloid β‐rich aggregate. Previous studies have indicated that PrP interacts with Alzheimer′s disease amyloid‐β peptide (Aβ), but it remains elusive how this interaction impacts on the misfolding of PrP. This study presents the first in vitro evidence that Aβ induces PrP‐amyloid formation at submicromolar concentrations. Interestingly, systematic mutagenesis of PrP revealed that Aβ requires no specific amino acid sequences in PrP, and induces the misfolding of other unrelated proteins (insulin and lysozyme) into amyloid fibrils in a manner analogous to PrP. This unanticipated nonspecific amyloidogenic effect of Aβ indicates that this peptide might be involved in widespread protein aggregation, regardless of the amino acid sequences of target proteins, and exacerbate the pathology of many neurodegenerative diseases.     

Use of non-aqueous capillary electrophoresis for the quality control of commercial saffron samples

A non-aqueous capillary electrophoresis (NACE) method for quantifying the seven crocin metabolites that are the major biologically active ingredients of saffron was developed. Separation is done by using a fused silica capillary filled with a 12.5 mM H3BO3/37.5 mM sodium tetraborate methanolic solution as background electrolyte. The results obtained were compared with the total index "safranal value", widely used as a quality measure of saffron products. The comparison revealed that the proposed NACE method provides useful information not obtained in the safranal value. Infact, samples with a similar safranal value can contain crocin metabolites in different concentrations and relative proportions. This new method is very useful for quality control in commercial saffron samples.     

Hydroxy-Porphyrin as an Effective,Endogenous Molecular Clamp during Early Stages of Amyloid Fibrillization

Amyloid fibril formation of proteins is of great concern in neurodegenerative disease and can be detrimental to the storage and stability of biologics. Recent evidence suggests that insulin fibril formation reduces the efficacy of type II diabetes management and may lead to several complications. To develop anti-amyloidogenic compounds of endogenous origin, we have utilized the hydrogen bond anchoring, π stacking ability of porphyrin, and investigated its role on the inhibition of insulin amyloid formation. We report that hydroxylation and metal removal from the heme moiety yields an excellent inhibitor of insulin fibril formation. Thioflavin T, tyrosine fluorescence, Circular Dichorism (CD) spectroscopy, Field emission scanning electron microscopy (FESEM) and molecular dynamics (MD) simulation studies suggest that hematoporphyrin (HP) having hydrogen bonding ability on both sides is a superior inhibitor compared to hemin and protoporphyrin (PP). Experiments with hen egg white lysozyme (HEWL) amyloid fibril formation also validated the efficacy of endogenous porphyrin based small molecules. Our results will help to decipher a general therapeutic strategy to counter amyloidogenesis.     

Interaction of biological molecules with clay minerals: a combined spectroscopic and sorption study of lysozyme on saponite

The interaction of hen egg white lysozyme (HEWL) with Na- and Cs-exchanged saponite was investigated using sorption, structural, and spectroscopic methods as a model system to study clay-protein interactions. HEWL sorption to Na- and Cs-saponite was determined using the bicinchoninic acid (BCA) assay, thermogravimetric analysis, and C and N analysis. For Na-saponite, the TGA and elemental analysis-derived sorption maximum was 600 mg/g corresponding to a surface coverage of 0.85 ng/mm(2) with HEWL occupying 526 m(2)/g based on a cross-sectional area of 13.5 nm(2)/molecule. HEWL sorption on Na-saponite was accompanied by the release of 9.5 Na(+) ions for every molecule of HEWL sorbed consistent with an ion exchange mechanism between the positively charged HEWL (IEP 11) and the negatively charged saponite surface. The d-spacing of the HEWL-Na-saponite complex increased to a value of 4.4 nm consistent with the crystallographic dimensions of HEWL of 3 × 3 × 4.5 nm. In the case of Cs-saponite, there was no evidence of interlayer sorption; however, sorption of HEWL to the "external" surface of Cs-saponite showed a high affinity isotherm. FTIR and Raman analysis of the amide I region of the HEWL-saponite films prepared from water and D(2)O showed little perturbation to the secondary structure of the protein. The overall hydrophilic nature of the HEWL-Na-saponite complex was determined by water vapor sorption measurements. The clay retained its hydrophilic character with a water content of 18% at high humidity corresponding to 240 H(2)O molecules per molecule of HEWL.     

Molecular Dynamics Simulation of Ester‐Linked Hen Egg White Lysozyme Reveals the Effect of Missing Backbone Hydrogen Bond Donors on the Protein Structure

The three‐dimensional structure of a protein is stabilized by a number of different atomic interactions. One of these is hydrogen bonding. Its influence on the spatial structure of the hen egg white lysozyme is investigated by replacing peptide bonds (except those of the two proline residues) by ester bonds. Molecular dynamics simulations of native and ester‐linked lysozyme are compared with the native crystal structure and with NOE distance bounds derived from solution NMR experiments. The ester‐linked protein shows a slight compaction while losing its native structure. However, it does not unfold completely. The structure remains compact due to its hydrophobic core and a changed network of hydrogen bonds involving side chains.     

Properties of a water layer on hydrophilic and hydrophobic self-assembled monolayer surfaces: A molecular dynamics study

The microscopic behaviors of a water layer on different hydrophilic and hydrophobic surfaces of well ordered self-assembled monolayers (SAMs) are studied by molecular dynamics simulations. The SAMs consist of 18-carbon alkyl chains bound to a silicon(111) substrate, and the characteristic of its surface is tuned from hydrophobic to hydrophilic by using different terminal functional groups ( CH 3 , COOH). In the simulation, the properties of water membranes adjacent to the surfaces of SAMs were reported by comparing pure water in mobility, structure, and orientational ordering of water molecules. The results suggest that the mobility of water molecules adjacent to hydrophilic surface becomes weaker and the molecules have a better ordering. The distribution of hydrogen bonds indicates that the number of water-water hydrogen bonds per water molecule tends to be lower. However, the mobility of water molecules and distribution of hydrogen bonds of a water membrane in hydropho- bic system are nearly the same as those in pure water system. In addition, hydrogen bonds are mainly formed between the hydroxyl of the COOH group and water molecules in a hydrophilic system, which is helpful in understanding the structure of interfacial water.     

The organization of aromatic side groups in an amyloid fibril probed by solid-state 2H and 19F NMR spectroscopy

Some 25 diseases are associated with proteins and peptides that assemble into amyloid fibrils composed of beta-strands connected by hydrogen bonds oriented parallel to the fiber long axis. There is mounting evidence that amyloid formation involves specific interactions between amino acid side groups, which bring together beta-sheets to form layers with buried and exposed faces. This work demonstrates how a combination of solid-state 2H and 19F NMR experiments can provide constraints on fibril architecture by probing the environment and spatial organisation of aromatic side groups. It is shown that phenylalanine rings within fibrils formed by a decapeptide fragment of the islet amyloid polypeptide, amylin, are highly motionally restrained and are situated within 6.5 A of one another. Taken together with existing structural constraints for this peptide, these results are consistent with a fibril architecture that comprises layers of two or more beta-sheets, with the aromatic residues facing into the inter-sheet space and possibly engaged in pi-pi interactions. The methods presented will be of general utility in exploring the architecture of fibrils of larger, full-length peptides and proteins, including amylin itself.     

Novel surface modified molecularly imprinted polymer using acryloyl-β-cyclodextrin and acrylamide as monomers for selective recognition of lysozyme in aqueous solution

A novel protein imprinted polymer for selective recognition of lysozyme was obtained. Acryloyl-β-cyclodextrin, which offered hydrophilic exterior and hydrophobic cavity that were allowed to self-assemble with the template protein through hydrogen interaction and hydrophobic interaction, was synthesized and used as the functional monomer. Polymerization was carried out in the presence of acrylamide as an assistant monomer, which resulted in a new type of protein imprinted polymer. Langmuir adsorption model was employed to describe the isotherms, and maximum adsorption capacity was evaluated. The performance of such imprinted polymer was further demonstrated by high-performance liquid chromatography, and the results showed that the column packed with the lysozyme imprinted silica beads could effectively separate lysozyme from the mixture of lysozyme–cytochrome c, lysozyme–bovine serum albumin, lysozyme–avidin or lysozyme–methylated bovine serum albumin, which showed its high selectivity.     

A new angle on heat capacity changes in hydrophobic solvation

The differential solubility of polar and apolar groups in water is important for the self-assembly of globular proteins, lipid membranes, nucleic acids, and other specific biological structures through hydrophobic and hydrophilic effects. The increase in water's heat capacity upon hydration of apolar compounds is one signature of the hydrophobic effect and differentiates it from the hydration of polar compounds, which cause a decrease in heat capacity. Water structuring around apolar and polar groups is an important factor in their differential solubility and heat capacity effects. Here, it is shown that joint radial/angular distribution functions of water obtained from simulations reveal quite different hydration structures around polar and apolar groups: polar and apolar groups have a deficit or excess, respectively, of "low angle hydrogen bonds". Low angle hydrogen bonds have a larger energy fluctuation than high angle bonds, and analysis of these differences provides a physical reason for the opposite changes in heat capacity and new insight into water structure around solutes and the hydrophobic effect.     

Controlling the morphology of cross beta-sheet assemblies by rational design

Low molecular weight peptidomimetics with simple amphiphilic sequences can help to elucidate the structures of cross beta-sheet assemblies, such as amyloid fibrils. The peptidomimetics described herein comprise a dibenzofuran template, two peptide strands made up of alternating hydrophilic and hydrophobic residues, and carboxyl termini, each of which can be varied to probe the structural requirements for beta-sheet self-assembly processes. The dibenzofuran template positions the strands approximately 10 A apart, allowing corresponding hydrophobic side chains in the strands to pack into a collapsed U-shaped structure. This conformation is stabilized by hydrophobic interactions, not intramolecular hydrogen bonds. Intermolecular stacking of the collapsed peptidomimetics, enabled by intermolecular hydrogen bonding and hydrophobic interactions, affords 25-27 A wide protofilaments having a cross beta-sheet structure. Association of protofilaments, mediated by the dibenzofuran substructures and driven by the hydrophobic effect, affords 50-60 A wide filaments. These widths can be controlled by changing the length of the peptide strands. Further assembly of the filaments into fibrils or ribbons can be controlled by modification of the template, C-terminus, and buffer ion composition.     

Molecular aggregates in aqueous solutions of bile acid salts. Molecular dynamics simulation study

The aggregation behavior of two bile acid salts (i.e., sodium cholate and sodium deoxycholate) has been studied in their aqueous solutions of three different concentrations (i.e., 30, 90,and 300 mM) by means of molecular dynamics computer simulations. To let the systems reach thermodynamic equilibrium, rather long simulations have been performed: the equilibration period, lasting for 20-50 ns, has been followed by a 20 ns long production phase, during which the average size of the bile aggregates (regarded to be the slowest varying observable) has already fluctuated around a constant value. The production phase of the runs has been about an order of magnitude longer than the average lifetime of both the monomeric bile ions and the bonds that link two neighboring bile ions together to be part of the same aggregate. This has allowed the bile ions belonging to various aggregates to be in a dynamic equilibrium with the isolated monomers. The observed aggregation behavior of the studied bile ions has been found to be in good qualitative agreement with experimental findings. The analysis of the results has revealed that, due to their molecular structure, which is markedly different from that of the ordinary aliphatic surfactants, the bile ions form rather different aggregates than the usual spherical micelles. In the lowest concentration solution studied, the bile ions only form small oligomers. In the case of deoxycholate, these oligomers, such as the ordinary micelles, are kept together by hydrophobic interactions, whereas in the sodium cholate system, small hydrogen-bonded aggregates (mostly dimers) are also present. In the highest concentration systems, the bile ions form large secondary micelles, which are kept together both by hydrophobic interactions and by hydrogen bonds. Namely, in these secondary micelles, small hydrophobic primary micelles are linked together via the formation of hydrogen bonds between their hydrophilic outer surfaces.     

Elongation of ordered peptide aggregate of an amyloidogenic hexapeptide NFGAIL observed in molecular dynamics simulations with explicit solvent

The mechanisms by which amyloidogenic peptides and proteins form soluble toxic oligomers remain elusive. We have studied the formation of partially ordered tetramers and well-ordered octamers of an amyloidogenic hexapeptide NFGAIL (residues 22-27 of the human islet amyloid polypeptide) in our previous work. Continuing the effort, we here probe the beta-sheet elongation process by a combined total of 2.0 micros molecular dynamics simulations with explicit solvent. In a set of 10 simulations with the peptides restrained to the extended conformation, we observed that the main growth mode was elongation along the beta-sheet hydrogen bonds through primarily a two-stage process. Driven by hydrophobic forces, the peptides initially attached to the surface of the ordered oligomer, moved quickly to the beta-sheet edges, and formed stable beta-sheet hydrogen bonds. Addition of peptides to the existing oligomer notably improved the order of the peptide aggregate in which labile outer layer beta-sheets were stabilized, which provides good templates for further elongation. These simulations suggested that elongation along the beta-sheet hydrogen bonds occurs at the intermediate stage when low-weight oligomers start to form. We did not observe significant preference toward either parallel or antiparallel beta-sheets at the elongation stage for this peptide. In another set of 10 unrestrained simulations, the dominant growth mode was disordered aggregation. Taken together, these results offered a glimpse at the molecular events leading to the formation of ordered and disordered low-weight oligomers.     

Adsorption Behavior of Lysozyme and Tween 80 at Hydrophilic and Hydrophobic Silica–Water Interfaces

Nonionic surfactants such as Tween 80 are used commercially to minimize protein loss through adsorption and aggregation and preserve native structure and activity. However, the specific mechanisms underlying Tween action in this context are not well understood. Here, we describe the interaction of the well-characterized, globular protein lysozyme with Tween 80 at solid–water interfaces. Hydrophilic and silanized, hydrophobic silica surfaces were used as substrates for protein and surfactant adsorption, which was monitored in situ, with ellipsometry. The method of lysozyme and Tween introduction to the surfaces was varied in order to identify the separate roles of protein, surfactant, and the protein–surfactant complex in the observed interfacial behavior. At the hydrophobic surface, the presence of Tween in the protein solution resulted in a reduction in amount of protein adsorbed, while lysozyme adsorption at the hydrophilic surface was entirely unaffected by the presence of Tween. In addition, while a Tween pre-coat prevented lysozyme adsorption on the hydrophobic surface, such a pre-coat was completely ineffective in reducing adsorption on the hydrophilic surface. These observations were attributed to surface-dependent differences in Tween binding strength and emphasize the importance of the direct interaction between surfactant and solid surface relative to surfactant–protein association in solution in the modulation of protein adsorption by Tween 80.     

Adsorption of amyloid beta-peptide at polymer surfaces: a neutron reflectivity study.

The adsorption of amyloid beta-peptide at hydrophilic and hydrophobic modified silicon-liquid interfaces was characterized by neutron reflectometry. Distinct polymeric films were used to obtain noncharged (Formvar), negatively (sodium poly(styrene sulfonate)) and positively charged (poly(allylamine hydrochloride)) hydrophilic as well as hydrophobic surfaces (polystyrene and a polysiloxane-dodecanoic acid complex). Amyloid beta-peptide was found to adsorb at positively charged hydrophilic and hydrophobic surfaces, whereas no adsorbed layer was detected on hydrophilic noncharged and negatively charged films. The peptide adsorbed at the positively charged film as patches, which were dispersed on the surface, whereas a uniform layer was observed at hydrophobic surfaces. The thickness of the adsorbed peptide layer was estimated to be approximately 20 A. The peptide formed a tightly packed layer, which did not contain water. These studies provide information about the affinity of the amyloid beta-peptide to different substrates in aqueous solution and suggest that the amyloid fibril formation may be driven by interactions with surfaces.     

Effect of amidoalkyl group as spacer on aggregation properties of guanidine-type surfactants

Dodecanoyl amidoalkylguanidine hydrochlorides (C(12)A(m)G, m = 2, 3, 4, 6) are cationic surfactants that have an amidoalkyl group (A(m)) as spacer between the cationic guanidine and hydrophobic groups in the molecule. The effect of the A(m) group on the aggregation properties of the surfactants was evaluated through measurements of their critical micelle concentration (cmc) value, Krafft point, phase behavior, area occupied by one molecule at the air/water interface, and micellar aggregation number. Dodecylguanidine hydrochloride (C(12)A(0)G) with no A(m) group is a unique cationic surfactant because it exhibits a strong tendency for self-assembly when compared with common ionic surfactants, due to the hydrogen bonding between its guanidine groups in addition to the hydrophobic interaction between its alkyl chains [M. Miyake, K. Yamada, N. Oyama, Langmuir 24 (2008) 8527-8532]. In contrast, C(12)A(m)G showed a decreasing tendency for self-assembly with increasing alkyl chain length, m, of the A(m) group up to m = 3, above which the tendency increased. Such changes in aggregation tendency of the surfactants were suggested to arise from an increased bulkiness of the hydrophilic part caused by the A(m) group, resulting in a decrease in the hydrogen bonding between the guanidine groups and an increase in micellization through the cooperative hydrophobic interaction between the hydrophilic groups. From the balance of these effects, the area of the hydrophilic part of C(12)A(4)G was the largest and the hydrogen bonding between the guanidine groups in C(12)A(4)G was weakened. It is suggested in guanidine-type surfactant that A(4) gave a similar aggregation tendency to traditional ionic surfactants and a weak effect for skin.