Insulin Complexation with Cyclodextrins—A Molecular Modeling Approach

Around 5% of the population of the world is affected with the disease called diabetes mellitus. The main medication of the diabetes is the insulin; the active form is the insulin monomer, which is an instable molecule, because the long storage time, or the high temperature, can cause the monomer insulin to adapt an alternative fold, rich in β-sheets, which is pharmaceutically inactive. The aim of this study is to form different insulin complexes with all the cyclodextrin used for pharmaceutical excipients (native cyclodextrin, methyl, hydroxyethyl, hydroxypropyl and sulfobutylether substituted β-cyclodextrin), in silico condition, with the AutoDock molecular modeling program, to determine the best type of cyclodextrin or cyclodextrin derivate to form a complex with an insulin monomer, to predict the molar ratio, the conformation of the complex, and the intermolecular hydrogen bonds formed between the cyclodextrin and the insulin. From the results calculated by the AutoDock program it can be predicted that insulin can make a stable complex with 5–7 molecules of hydroxypropyl-β-cyclodextrin or sulfobutylether-β-cyclodextrin, and by forming a complex potentially can prevent or delay the amyloid fibrillation of the insulin and increase the stability of the molecule.

The Cryo-EM Structures of two Amphibian Antimicrobial Cross-β Amyloid Fibrils

The amyloid-antimicrobial link hypothesis is based on antimicrobial properties found in human amyloids involved in neurodegenerative and systemic diseases, along with amyloidal structural properties found in antimicrobial peptides (AMPs) across kingdoms of life. Supporting this hypothesis, we here determined the fibril structure of two AMPs from amphibians, uperin 3.5 and aurein 3.3, by cryogenic electron microscopy (cryo-EM), revealing amyloid cross-β fibrils of mated β-sheets at atomic resolution. Uperin 3.5 displayed substantial polymorphism with a protofilament of two mated β-sheets. The determined structure was a polymorph showing a 3-blade symmetrical propeller of nine peptides per fibril layer including tight β-sheet interfaces. This cross-β cryo-EM structure complements the cross-α fibril conformation previously determined by a crystal structure, substantiating a secondary structure switch mechanism of uperin 3.5. The aurein 3.3 arrangement consisted of six peptides per fibril layer, all showing kinked β-sheets allowing a rounded compactness of the fibril. The kinked β-sheets are similar to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. The amyloidal properties of antimicrobial peptides shed light on a mechanism of regulation of animicrobial activity involving self-assembly and fibril morphological variations. Moreover, the known endurance of amyloid structures can provide a template for the design of sturdy antimicrobials.

2.2 CuAAC in Peptidomimetics and Protein Mimics

The tremendous recent developments in click chemistry, including the impressive developments of strain-promoted cycloaddition reagents, all started with the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction conceived by Meldal et al. and Sharpless et al. This led to a revolution of extremely important applications in the chemical, biological, medical, and materials sciences. It is fair to state that, especially in the synthesis of multifunctional and complex small-to-large biomolecular constructs, CuAAC has been indispensable. This has been particularly evident in the area of peptides, peptidomimetics, and protein mimics. These biomolecules play key roles in the various peptide–peptide, peptide–protein, and protein–protein interactions that are involved in many diseases and disorders, and peptide-based therapeutics can be important in this context. However, it is often important to improve the bioactivity and overall stability, and modulate the spatial structure, of peptide-based therapeutics. The incorporation of the 1,4-disubstituted 1,2,3-triazole moiety as a non-native structural element using CuAAC is explored in this chapter. The resulting incorporated triazole moiety can lead to structural surrogates of the amide bond and disulfide bond. As a consequence, CuAAC can be utilized toward introducing conformational constraints and stabilizing secondary structures of α-helices, β-sheets/turns, or loop-like structures. In addition, CuAAC can be used to combine various peptide sequences with molecular scaffolds to develop protein mimics that can find applications as synthetic vaccines and antibodies.

Label-free characterisation of amyloids and alpha-Synuclein polymorphs by exploiting their intrinsic fluorescence property

Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime represented by model-free phasor plots, as a label-free assay to characterise amyloid structure. Intrinsic amyloid fluorescence arises from structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids (i.e., α-Synuclein (αS), β-Lactoglobulin and TasA) and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of pre-formed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct to those of their fibrillar counterpart. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathology), and for testing small molecule inhibitory compounds.

Cryo-EM structures of τ filaments from human brain

Electron cryo-microscopy (cryo-EM) has made it possible to determine near-atomic structures of τ filaments from human brain. Previous work had shown that the cores of paired helical and straight filaments of Alzheimer’s disease are made of two identical, but differently arranged C-shaped protofilaments. In recent years, cryo-EM has shown that the Alzheimer τ fold is 79 amino acids long. Five of the eight β-strands give rise to two antiparallel β-sheets, with the other three forming a β-helix. High-affinity binding sites of positron emission tomography ligand APN-1607 (PM-PBB3) are in the β-helix region. The Alzheimer fold contrasts with the 94 amino acid-long Pick fold, which is J-shaped and comprises nine β-strands that give rise to four antiparallel β-sheets, in the absence of a β-helix. Chronic traumatic encephalopathy τ fold is similar to the Alzheimer fold, but differs in the β-helix region, which is larger and contains a non-proteinaceous density that is probably hydrophobic. These folds are mostly two-layered. By contrast, the 107 amino acid τ fold of the 4R tauopathy corticobasal degeneration is four-layered and comprises 11 β-strands. It contains an internal, probably hydrophilic, density that is surrounded by τ. The τ folds described here share the presence of microtubule-binding repeats 3 and 4, as well as 10–13 amino acids after repeat 4.

Revealing Topological Barriers against Knot Untying in Thermal and Mechanical Protein Unfolding by Molecular Dynamics Simulations

The knot is one of the most remarkable topological features identified in an increasing number of proteins with important functions. However, little is known about how the knot is formed during protein folding, and untied or maintained in protein unfolding. By means of all-atom molecular dynamics simulation, here we employ methyltransferase YbeA as the knotted protein model to analyze changes of the knotted conformation coupled with protein unfolding under thermal and mechanical denaturing conditions. Our results show that the trefoil knot in YbeA is occasionally untied via knot loosening rather than sliding under enhanced thermal fluctuations. Through correlating protein unfolding with changes in the knot position and size, several aspects of barriers that jointly suppress knot untying are revealed. In particular, protein unfolding is always prior to knot untying and starts preferentially from separation of two α-helices (α1 and α5), which protect the hydrophobic core consisting of β-sheets (β1–β4) from exposure to water. These β-sheets form a loop through which α5 is threaded to form the knot. Hydrophobic and hydrogen bonding interactions inside the core stabilize the loop against loosening. In addition, residues at N-terminal of α5 define a rigid turning to impede α5 from sliding out of the loop. Site mutations are designed to specifically eliminate these barriers, and easier knot untying is achieved under the same denaturing conditions. These results provide new molecular level insights into the folding/unfolding of knotted proteins.

Synthesis, Characterization and Structure Properties of Biobased Hybrid Copolymers Consisting of Polydiene and Polypeptide Segments

Novel hybrid materials of the PB-b-P(o-Bn-L-Tyr) and PI-b-P(o-Bn-L-Tyr) type (where PB: 1,4/1,2-poly(butadiene), PI:3,4/1,2/1,4-poly(isoprene) and P(o-Bn-L-Tyr):poly(ortho-benzyl-L-tyrosine)) were synthesized through anionic and ring-opening polymerization under high-vacuum techniques. All final materials were molecularly characterized through infrared spectroscopy (IR) and proton and carbon nuclear magnetic resonance (1H-NMR, 13C-NMR) in order to confirm the successful synthesis and the polydiene microstructure content. The stereochemical behavior of secondary structures (α-helices and β-sheets) of the polypeptide segments combined with the different polydiene microstructures was also studied. The influence of the α-helices and β-sheets, as well as the polydiene chain conformations on the thermal properties (glass transition temperatures, thermal stability, α- and β-relaxation) of the present biobased hybrid copolymers, was investigated through differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and dielectric spectroscopy (DS). The obtained morphologies in thin films for all the synthesized materials via atomic force microscopy (AFM) indicated the formation of polypeptide fibrils in the polydiene matrix.

Kinetic interplay between droplet maturation and coalescence modulates shape of aged protein condensates

Biomolecular condensates formed by the process of liquid–liquid phase separation (LLPS) play diverse roles inside cells, from spatiotemporal compartmentalisation to speeding up chemical reactions. Upon maturation, the liquid-like properties of condensates, which underpin their functions, are gradually lost, eventually giving rise to solid-like states with potential pathological implications. Enhancement of inter-protein interactions is one of the main mechanisms suggested to trigger the formation of solid-like condensates. To gain a molecular-level understanding of how the accumulation of stronger interactions among proteins inside condensates affect the kinetic and thermodynamic properties of biomolecular condensates, and their shapes over time, we develop a tailored coarse-grained model of proteins that transition from establishing weak to stronger inter-protein interactions inside condensates. Our simulations reveal that the fast accumulation of strongly binding proteins during the nucleation and growth stages of condensate formation results in aspherical solid-like condensates. In contrast, when strong inter-protein interactions appear only after the equilibrium condensate has been formed, or when they accumulate slowly over time, with respect to the time needed for droplets to fuse and grow, spherical solid-like droplets emerge. By conducting atomistic potential-of-mean-force simulations of NUP-98 peptides —prone to forming inter-protein β-sheets— we observe that formation of inter-peptide β-sheets increases the strength of the interactions consistently with the loss of liquid-like condensate properties we observe at the coarse-grained level. Overall, our work aids in elucidating fundamental molecular, kinetic, and thermodynamic mechanisms linking the rate of change in protein interaction strength to condensate shape and maturation during ageing.