The physics of protein self-assembly

Understanding protein self-assembly is important for many biological and industrial processes. Proteins can self-assemble into crystals, filaments, gels, and other amorphous aggregates. The final forms include virus capsids and condensed phases associated with diseases such as amyloid fibrils. Although seemingly different, these assemblies all originate from fundamental protein interactions and are driven by similar thermodynamic and kinetic factors. Here we review recent advances in understanding protein self-assembly through a soft condensed matter perspective with an emphasis on three specific systems: globular proteins, viruses, and amyloid fibrils. We conclude with a discussion of unanswered questions in the field.     

Cytochrome display on amyloid fibrils

Protein amyloid fibrils can be functionalized by coating the core protofilament with high concentrations of proteins and enzymes. This can be done elegantly by appending a functional domain to an amyloidogenic protein monomer, then assembling the monomers into a fibril. To display an array of biologically functional porphyrins on the surface of protein fibrils, we have fused the sequence of the small, soluble cytochrome b562 to an SH3 dimer sequence that can form classical amyloid fibrils rapidly under well-defined conditions. The resulting fusion protein also forms amyloid fibrils and, in addition, binds metalloporphyrins, at half of the porphyrin binding sites as shown by UV-vis and NMR spectroscopies. Once metalloporphyrins are bound to the fibrils, the resulting holo-cytochrome domains are spectroscopically identical to the wild type cytochrome. The concentration of metalloporphyrins on a saturated fibril is estimated to be of the order of approximately 20 mM, suggesting that they could be interesting systems for applications in nanotechnology.     

Near-Wall Aggregation of Amyloidogenic Aβ 1-40 Peptide: Direct Observation by the FRET

The formation of amyloid fibrils is one of the variants of the self-organization of polypeptide chains. For the amyloid aggregation, the solution must be oversaturated with proteins. The interface of the liquid (solution) and solid (vessel walls) phases can trigger the adsorption of protein molecules, and the resulting oversaturation can initiate conformational transitions in them. In any laboratory experiment, we cannot exclude the presence of surfaces such as the walls of vessels, cuvettes, etc. However, in many works devoted to the study of amyloid formation, this feature is not considered. In our work, we investigated the behavior of the Aβ 1-40 peptide at the water–glass, water–quartz, and water–plastic interface. We carried out a series of simple experiments and showed that the Aβ 1-40 peptide is actively adsorbed on these surfaces, which leads to a significant interaction and aggregation of peptides. This means that the interface can be the place where the first amyloid nucleus appears. We suggest that this effect may also be one of the reasons for the difficulty of reproducing kinetic data when studying the aggregation of the amyloid of the Aβ 1-40 peptide and other amyloidogenic proteins     

Pathway Dependence of the Formation and Development of Prefibrillar Aggregates in Insulin B Chain

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.     

Significance of Oligomeric and Fibrillar Species in Amyloidosis: Insights into Pathophysiology and Treatment

Amyloidosis is a term referring to a group of various protein-misfolding diseases wherein normally soluble proteins form aggregates as insoluble amyloid fibrils. How, or whether, amyloid fibrils contribute to tissue damage in amyloidosis has been the topic of debate. In vitro studies have demonstrated the appearance of small globular oligomeric species during the incubation of amyloid beta peptide (Aβ). Nerve biopsy specimens from patients with systemic amyloidosis have suggested that globular structures similar to Aβ oligomers were generated from amorphous electron-dense materials and later developed into mature amyloid fibrils. Schwann cells adjacent to amyloid fibrils become atrophic and degenerative, suggesting that the direct tissue damage induced by amyloid fibrils plays an important role in systemic amyloidosis. In contrast, there is increasing evidence that oligomers, rather than amyloid fibrils, are responsible for cell death in neurodegenerative diseases, particularly Alzheimer’s disease. Disease-modifying therapies based on the pathophysiology of amyloidosis have now become available. Aducanumab, a human monoclonal antibody against the aggregated form of Aβ, was recently approved for Alzheimer’s disease, and other monoclonal antibodies, including gantenerumab, solanezumab, and lecanemab, could also be up for approval. As many other agents for amyloidosis will be developed in the future, studies to develop sensitive clinical scales for identifying improvement and markers that can act as surrogates for clinical scales should be conducted.     

Inhibition of Amyloid Fibril Growth and Dissolution of Amyloid Fibrils by Curcumin–Gold Nanoparticles

Inhibition of amyloid fibrillation and clearance of amyloid fibrils/plaques are essential for the prevention and treatment of various neurodegenerative disorders involving protein aggregation. Herein, we report curcumin‐functionalized gold nanoparticles (Au‐curcumin) of hydrodynamic diameter 10–25 nm, which serve to inhibit amyloid fibrillation and disintegrate/dissolve amyloid fibrils. In nanoparticle form, curcumin is water‐soluble and can efficiently interact with amyloid protein/peptide, offering enhanced performance in inhibiting amyloid fibrillation and dissolving amyloid fibrils. Our results imply that nanoparticle‐based artificial molecular chaperones may offer a promising therapeutic approach to combat neurodegenerative disease.     

Inhibition of amyloid aggregation by formation of helical assemblies

The formation of amyloid aggregates is responsible for a wide range of diseases, including Alzheimer's and Parkinson's disease. Although the amyloid-forming proteins have different structures and sequences, all undergo a conformational change to form amyloid aggregates that have a characteristic cross-β-structure. The mechanistic details of this process are poorly understood, but different strategies for the development of inhibitors of amyloid formation have been proposed. In most cases, chemically diverse compounds bind to an elongated form of the protein in a β-strand conformation and thereby exert their therapeutic effect. However, this approach could favor the formation of prefibrillar oligomeric species, which are thought to be toxic. Herein, we report an alternative approach in which a helical coiled-coil-based inhibitor peptide has been designed to engage a coiled-coil-based amyloid-forming model peptide in a stable coiled-coil arrangement, thereby preventing rearrangement into a β-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, we show that the helix-forming peptide is able to disassemble mature amyloid-like fibrils.     

生物液晶

液晶是一种介于液相和固相之间的中间相，具有流动性和有序性，其性质表明它是一种极适于生命特征的状态。生命体中的蛋白质、核酸、多糖、脂类等都能够通过自组装而呈现液晶态，其液晶行为与细胞和组织功能的表达有关。本文介绍了液晶的分类、表征方法及生命体内的蛋白质、脱氧核糖核酸、多糖、脂类的液晶特性以及液晶态的生物材料与细胞的相互作用。     

酚化合物对胰岛素淀粉样纤维化的抑制作用

采用牛胰岛素作为模型多肽分子, 对几种结构相近的简单多酚的抗多肽淀粉样纤维化作用进行了研究. 结果表明, 邻苯二酚和对苯二酚对胰岛素纤维化具有抑制作用, 并通过形成醌中间体对多肽链进行修饰, 与对苯醌作用类似; 而苯酚和间二苯酚在相同条件下, 既不能修饰多肽也无抑制纤维化作用. 在无氧条件下, 邻苯二酚和对苯二酚对胰岛素纤维化的抑制作用明显降低, 说明酚化合物经氧化形成的醌中间体是其抗胰岛素纤维化的主要活性结构.     

Self-assembly and morphological characterization of two-component functional amyloid proteins

Functional amyloid has been increasingly applied as self-assembling nanostructures to construct multifunctional biomaterials. However, little has been known how different side domains, varied fusion positions and subunits affect self-assembly and morphologies of amyloid fibrils. Here, we constructed three groups of two-component amyloid proteins based on CsgA, the major protein components of Escherichia coli biofilms, to bridge these gaps. We showed that all fusion proteins have amyloid features, as indicated by Congo red assay. Atomic force microscopy (AFM) indeed reveals that these fusion proteins are able to self-assemble into fibrils, with an average diameter of 0.5-2 nm and length of hundreds of nanometers to several micrometers. The diameter of fibrils increases with the increase of the molecular weight of fusion domains, while the dynamic assembly of recombinant proteins was delayed as a result of the introduction of fusion domains. Moreover, fusion of the same functional domains but at intermediate position seems to cause the most interference on fibril assembly compared with those fused at C or Nterminus, as mainly short and irregular fibrils were detected. This phenomenon appears more pronounced for randomly coiled mussel foot proteins (Mfps) than for rigid chitin-binding domain (CBD). Finally, increase of the molecular weight of tandem repeats in protein monomer seemed to increase the fibril diameter of the resultant fibrils, but either reduction of the tandem repeats of CsgA to one single belta-sheet loop or increase in the number of tandem repeats of CsgAs from one to four produced shorter and intermittent fibrils compared with CsgA control protein. These studies therefore provide insights into self-assembly of two-component amyloid proteins and lay the foundation for rational design of multifunctional molecular biomaterials.     

Structural insights into the polymorphism of amyloid-like fibrils formed by region 20-29 of amylin revealed by solid-state NMR and X-ray fiber diffraction

Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.     

Structural regulation of a peptide-conjugated graft copolymer: a simple model for amyloid formation

The self-assembly of peptides and proteins into beta-sheet-rich high-order structures has attracted much attention as a result of the characteristic nanostructure of these assemblies and because of their association with neurodegenerative diseases. Here we report the structural and conformational properties of a peptide-conjugated graft copolymer, poly(gamma-methyl-L-glutamate) grafted polyallylamine (1) in a water-2,2,2-trifluoroethanol solution as a simple model for amyloid formation. Atomic force microscopy revealed that the globular peptide 1 self-assembles into nonbranching fibrils that are about 4 nm in height under certain conditions. These fibrils are rich in beta-sheets and, similar to authentic amyloid fibrils, bind the amyloidophilic dye Congo red. The secondary and quaternary structures of the peptide 1 can be controlled by manipulating the pH, solution composition, and salt concentration; this indicates that the three-dimensional packing arrangement of peptide chains is the key factor for such fibril formation. Furthermore, the addition of carboxylic acid-terminated poly(ethylene glycol), which interacts with both of amino groups of 1 and hydrophobic PMLG chains, was found to obviously inhibit the alpha-to-beta structural transition for non-assembled peptide 1 and to partially cause a beta-to-alpha structural transition against the 1-assembly in the beta-sheet form. These findings demonstrate that the amyloid fibril formation is not restricted to specific protein sequences but rather is a generic property of peptides. The ability to control the assembled structure of the peptide should provide useful information not only for understanding the amyloid fibril formation, but also for developing novel peptide-based material with well-defined nanostructures.     

The amyloidogenicity of gelsolin is controlled by proteolysis and pH.

BACKGROUND: Normally, gelsolin functions in plasma as part of the actin-scavenging system to assemble and disassemble actin filaments. The Asp 187-->Asn (D187N) Asp 187-->Tyr (D187Y) gelsolin mutations facilitate two proteolytic cuts in the parent protein generating a 71-residue fragment that forms amyloid fibrils in humans, putatively causing Finnish type familial amyloidosis (FAF). We investigated the role of the D187N mutation in amyloidogenicity using biophysical studies in vitro. RESULTS: Both the recombinant wild-type and D187N FAF-associated gelsolin fragments adopt an ensemble of largely unfolded structures that do not self-associate into amyloid at pH 7. 5. Incubation of either fragment at low pHs (6.0-4.0) leads to the formation of well-defined fibrils within 72 hours, however. CONCLUSIONS: The D187N mutation has been suggested to destabilize the structure of the gelsolin parent protein (specifically domain 2), facilitating two proteolytic cleavage events. Our studies demonstrate that generating the largely unstructured peptide is not sufficient alone for amyloid formation in vitro (on a time scale of months). A drop in pH or an analogous environmental change appears necessary to convert the unstructured fragment into amyloid fibrils, probably through an associative mechanism. The wild-type gelsolin fragment will make amyloid fibrils from pH 6 to 4 in vitro, but neither the wild-type fragment nor fibrils have been observed in vivo. It is possible that domain 2 of wild-type gelsolin is stable in the context of the whole protein and not susceptible to the proteolytic degradation that affords the 71-residue FAF-associated peptide.     

Supramolecular hydrogen-bonded liquid crystals

The role of hydrogen-bonding interactions in the formation and/or stabilization of liquid crystalline phases has been recognized in recent years and significant work has been conducted. Following the first and well-established examples of liquid crystal formation through the dimerization of aromatic carboxylic acids, several classes of compounds have been prepared by the interaction of complementary molecules, the liquid crystalline behaviour of which is crucially dependent on the structure of the resulting supramolecular systems. In this review the main classes of liquid crystals prepared through hydrogen-bonding interactions are presented, with the aim of establishing, in the first place, the diversity of organic compounds that can be used as building elements in the process of liquid crystal formation. Rigid-rod anisotropic or amphiphilic-type molecules, appropriately functionalized with recognizable moieties, interact in the melt or in solution and lead to the formation of supramolecular complexes that may exhibit thermotropic liquid crystalline character. Depending on the nature, number and position of the groups able to form hydrogen bonds, a diversity of supramolecular structures, both dimeric and polymeric, have been obtained, affording in turn various liquid crystalline phases. The structure and stability of these hydrogen-bonded supramolecular complexes and their relation to the observed liquid crystalline phases are the main topics of this review.     

Supramolecular hydrogen-bonded liquid crystals

The role of hydrogen-bonding interactions in the formation and/or stabilization of liquid crystalline phases has been recognized in recent years and significant work has been conducted. Following the first and well-established examples of liquid crystal formation through the dimerization of aromatic carboxylic acids, several classes of compounds have been prepared by the interaction of complementary molecules, the liquid crystalline behaviour of which is crucially dependent on the structure of the resulting supramolecular systems. In this review the main classes of liquid crystals prepared through hydrogen-bonding interactions are presented, with the aim of establishing, in the first place, the diversity of organic compounds that can be used as building elements in the process of liquid crystal formation. Rigid-rod anisotropic or amphiphilic-type molecules, appropriately functionalized with recognizable moieties, interact in the melt or in solution and lead to the formation of supramolecular complexes that may exhibit thermotropic liquid crystalline character. Depending on the nature, number and position of the groups able to form hydrogen bonds, a diversity of supramolecular structures, both dimeric and polymeric, have been obtained, affording in turn various liquid crystalline phases. The structure and stability of these hydrogen-bonded supramolecular complexes and their relation to the observed liquid crystalline phases are the main topics of this review.     

Two-dimensional chiral model for liquid crystals, bent hard needles: a Monte Carlo simulation

The liquid crystalline behavior of a two dimensional (2D) model of hard needles bent into a "zigzag shape" is studied. This model, originally designed to study two dimensional chiral segregation, also shows liquid crystalline behavior and has some anomalous features which are contrasted in relation to the following: (i) Most of the microscopical models used to study liquid crystals have a symmetry axis that coincides with a molecular axis; (ii) in three-dimensions, chiral molecules can form cholesteric instead of nematic phases; (iii) the smectic phase is usually found when attractions are present or at least when the molecules have finite volume. Despite the fact that the present 2D model does not have any of these characteristics, numerical evidence is found for the occurrence of nematic and smectic phases. Since these molecules are athermal, infinitely repulsive, and infinitesimally thin, the liquid crystalline characteristics are attributed to excluded volume effects. To determine the mesophases of the model, both nematic and smectic order parameters as well as distribution functions are computed.     

Distribution of Amyloid‐Like and Oligomeric Species from Protein Aggregation Kinetics

Amyloid fibrils and soluble oligomers are two types of protein aggregates associated with neurodegeneration. Classic therapeutic strategies try to prevent the nucleation and spread of amyloid fibrils, whilst diffusible oligomers have emerged as promising drug targets affecting downstream pathogenic processes. We developed a generic protein aggregation model and validate it against measured compositions of fibrillar and non‐fibrillar assemblies of ataxin‐3, a protein implicated in Machado–Joseph disease. The derived analytic rate‐law equations can be used to 1) identify the presence of parallel aggregation pathways and 2) estimate the critical sizes of amyloid fibrils. The discretized population balance supporting our model is the first to quantitatively fit time‐resolved measurements of size and composition of both amyloid‐like and oligomeric species. The new theoretical framework can be used to screen a new class of drugs specifically targeting toxic oligomers.     

A designed protein interface that blocks fibril formation

Protein fibril formation is implicated in many diseases, and therefore much effort has been focused toward the development of inhibitors of this process. In a previous project, a monomeric protein was computationally engineered to bind itself and form a heterodimer complex following interfacial redesign. One of the protein monomers, termed monomer-B, was unintentionally destabilized and shown to form macroscopic fibrils. Interestingly, in the presence of the designed binding partner, fibril formation was blocked. Here we describe the complete characterization of the amyloid properties of monomer-B and the inhibition of fiber formation by the designed binding partner, monomer-A. Both proteins are mutants of the betal domain of streptococcal protein-G. The free monomer-B protein forms amyloid-type fibrils, as determined by transmission electron microscopy and the change in fluorescence of Thioflavin T, an amyloid-specific dye. Fibril formation kinetics are influenced by pH, protein concentration, and seeding with preformed fibrils. Under all conditions tested, monomer-A was able to inhibit the formation of monomer-B fibrils. This inhibition is specific to the engineered interaction, as incubation of monomer-B with wild-type protein-G (a structural homologue) did not result in inhibition under the same conditions. Thus, this de novo-designed heterodimeric complex is an excellent model system for the study of protein-based fibril formation and inhibition. This system provides additional insight into the development of pharmaceuticals for amyloid disorders, as well as the potential use of amyloid fibrils for self-assembling nanostructures.     

Silica Nanowires Templated by Amyloid‐like Fibrils

Many peptides self‐assemble to form amyloid fibrils. We previously explored the sequence propensity to form amyloid using variants of a designed peptide with sequence KFFEAAAKKFFE. These variant peptides form highly stable amyloid fibrils with varied lateral assembly and are ideal to template further assembly of non‐proteinaceous material. Herein, we show that the fibrils formed by peptide variants can be coated with a layer of silica to produce silica nanowires using tetraethyl‐orthosilicate. The resulting nanowires were characterized using electron microscopy (TEM), X‐ray fiber diffraction, FTIR and cross‐section EM to reveal a nanostructure with peptidic core. Lysine residues play a role in templating the formation of silica on the fibril surface and, using this library of peptides, we have explored the contributions of lysine as well as arginine to silica templating, and find that sequence plays an important role in determining the physical nature and structure of the resulting nanowires.     

Ordered 2-D and 3-D nanostructured amphiphile self-assembly materials stable in excess solvent

Amphiphile lyotropic liquid crystalline self-assembly materials are being used for a diverse range of applications. Historically, the most studied lyotropic liquid crystalline phase is probably the one-dimensional (1-D) lamellar phase, which has been employed as a model system for biomembranes and for drug delivery applications. In recent years, the structurally more complex 2-D and 3-D ordered lyotropic liquid crystalline phases, of which reversed hexagonal (H(2)) and reversed cubic phases (v(2)) are two prominent examples, have received growing interest. As is the case for the lamellar phase, these phases are frequently stable in excess water, which facilitates the preparation of nanoparticle dispersions and makes them suitable candidates for the encapsulation and controlled release of drugs. Integral membrane protein crystallization media and templates for the synthesis of inorganic nanostructured materials are other applications for 2-D and 3-D amphiphile self-assembly materials. The number of amphiphiles identified as forming nanostructured reversed phases stable in excess solvent is rapidly growing. In this article, different classes of amphiphiles that form reversed phases in excess solvent are reviewed, with an emphasis on linking phase behavior to amphiphile structure. The different amphiphile classes include: ethylene oxide-, monoacylglycerol-, glycolipid-, phosphatidylethanolamine-, and urea-based amphiphiles.