Tunable Self-Assembled Peptide Hydrogel Sensor for Pharma Cold Supply Chain.

Defrost sensors are a crucial element for proper functioning of the pharmaceutical cold chain. In this paper, the self-assembled peptide-based hydrogels were used to construct a sensitive defrost sensor for the transportation and storage of medications and biomaterials. The turbidity of the peptide hydrogel was employed as a marker of the temperature regime. The gelation kinetics under different conditions was studied to detect various stages of hydrogel structural transitions aimed at tuning the system properties. The developed sensor can be stored at room temperature for a long period, irreversibly indicates whether the product has been thawed, and can be adjusted to a specific temperature range and detection time.

On-off transition and ultrafast decay of amino acid luminescence driven by modulation of supramolecular packing.

Luminescence of biomolecules in the visible range of the spectrum has been experimentally observed upon aggregation, contrary to their monomeric state. However, the physical basis for this phenomenon is still elusive. Here, we systematically examine all coded amino acids to provide non-biased empirical insights. Several amino acids, including non-aromatic, show intense visible luminescence. Lysine crystals display the highest signal, whereas the very chemically similar non-coded ornithine does not, implying a role for molecular packing rather than the chemical characteristics. Furthermore, cysteine shows luminescence that is indeed crystal packing dependent as repeated rearrangements between two crystal structures result in a reversible on-off optical transition. In addition, ultrafast lifetime decay is experimentally validated, corroborating a recently raised hypothesis regarding the governing role of nπ∗ states in the emission formation. Collectively, our study supports that electronic interactions between non-fluorescent, non-absorbing molecules at the monomeric state may result in reversible optically active states by the formation of supramolecular fluorophores.

Transition of Metastable Cross-α Crystals into Cross-ß Fibrils by ß-Turn Flipping.

The ensemble of native, folded state was once considered to represent the global energy minimum of a given protein sequence. More recently, the discovery of the cross-ß amyloid state revealed that deeper energy minima exist, often associated with pathogenic, fibrillar deposits, when the concentration of proteins reaches a critical value. Fortunately, a sizable energy barrier impedes the conversion from native to pathogenic states. However, little is known about the structure of the related transition state. In addition, there are indications of polymorphism in the amyloidogenic process. Here, we report the first evidence of the conversion of metastable cross-α-helical crystals to thermodynamically stable cross-ß-sheet-like fibrils by a de novo designed heptapeptide. Furthermore, for the first time, we demonstrate at atomic resolution that the flip of a peptide plane from a type I to a type II' turn facilitates transformation to cross-ß structure and assembly of a dry steric zipper. This study establishes the potential of a peptide turn, a common protein secondary structure, to serve as a principal gatekeeper between a native metastable folded state and the amyloid state.

Intrinsic Fluorescence of Metabolite Amyloids Allows Label-Free Monitoring of Their Formation and Dynamics in Live Cells.

The formation of apoptosis-inducing amyloidal structures by metabolites has significantly extended the "amyloid hypothesis" to include non-proteinaceous, single metabolite building blocks. However, detection of metabolite assemblies is restricted compared to their larger protein-based counterparts owing to the hindrance of external labelling and limited immunohistochemical detection tools. Herein, we present the detection of the formation, dynamics, and cellular distribution of metabolite amyloid-like structures and provide mechanistic insights into the generation of supramolecular chromophores. Moreover, the intrinsic fluorescence properties allow the detection of metabolite assemblies in living cells without the use of external dyes. Altogether, this intrinsic fluorescence of metabolite assemblies further verifies their amyloidal nature, while providing an important tool for further investigation of their pathological role in inborn error of metabolism disorders.

Seeding of proteins into amyloid structures by metabolite assemblies may clarify certain unexplained epidemiological associations.

The accumulation of various metabolites appears to be associated with diverse human diseases. However, the aetiological link between metabolic alteration and the observed diseases is still elusive. This includes the correlation between the abnormally high levels of homocysteine and quinolinic acid in Alzheimer's disease, as well as the accumulation of oncometabolites in malignant processes. Here, we suggest and discuss a possible mechanistic insight into metabolite accumulation in conditions such as neurodegenerative diseases and cancer. Our hypothesis is based on the demonstrated ability of metabolites to form amyloid-like structures in inborn error of metabolism disorders and the potential of such metabolite amyloids to promote protein aggregation. This notion can provide a new paradigm for neurodegeneration and cancer, as both conditions were linked to loss of function due to protein aggregation. Similar to the well-established observation of amyloid formation in many degenerative disorders, the formation of amyloids by tumour-suppressor proteins, including p53, was demonstrated in malignant states. Moreover, this new paradigm could fill the gap in understanding the high occurrence of specific types of cancer among genetic error of metabolism patients. This hypothesis offers a fresh view on the aetiology of some of the most abundant human maladies and may redirect the efforts towards new therapeutic developments.

Bioinspired Flexible and Tough Layered Peptide Crystals.

One major challenge of functional material fabrication is combining flexibility, strength, and toughness. In several biological and artificial systems, these desired mechanical properties are achieved by hierarchical architectures and various forms of anisotropy, as found in bones and nacre. Here, it is reported that crystals of N-capped diphenylalanine, one of the most studied self-assembling systems in nanotechnology, exhibit well-ordered packing and diffraction of sub-Å resolution, yet display an exceptionally flexible nature. To explore this flexibility, the mechanical properties of individual crystals are evaluated, assisted by density functional theory calculations. High-resolution scanning electron microscopy reveals that the crystals are composed of layered self-assembled structures. The observed combination of strength, toughness, and flexibility can therefore be explained in terms of weak interactions between rigid layers. These crystals represent a novel class of self-assembled layered materials, which can be utilized for various technological applications, where a combination of usually contradictory mechanical properties is desired.

Diphenylalanine as a Reductionist Model for the Mechanistic Characterization of ß-Amyloid Modulators.

The phenomenon of protein aggregation into amyloid fibrils is associated with a large number of major diseases of unrelated etiology. Unraveling the mechanism of amyloid self-assembly and identifying therapeutic directions to control this process are of utmost importance. Research in this field has been hampered by several challenges, including reproducibility, low protein purification yields, and the inherent aggregation propensity of amyloidogenic proteins, making them extremely difficult to study. Herein, on the basis of the similarity in the assembly mechanism, as well as the physical, chemical, and biological characteristics, of diphenylalanine nanostructures and aromatic amino acid containing amyloid fibrils, we report a simple, yet robust peptide-based platform that could be used for screening of small molecules potentially capable of interfering with the aggregation process and for mechanistic exploration of their mode of action. The system was validated using four small-molecule inhibitors, and the effect was examined via turbidity assay, thioflavin T fluorescence, and electron microscopy. The aggregation profile of diphenylalanine was very similar to that of ß-amyloid polypeptide in the presence of the modulators. Rosmarinic acid emerged as an extremely potent inhibitor and a destabilizer of the aggregates. The effect of stoichiometric variation of rosmarinic acid on the process of destabilization was also probed using a microfluidic technique. Finally, the formation of equimolar complexes of diphenylalanine and inhibitors was detected using mass spectrometry. This approach not only provides a system for high-throughput screening of possible inhibitor molecules from larger libraries of modulators, but is also highly useful for understanding the mechanistic aspects of the interactions leading to the process of inhibition.

Dynamic microfluidic control of supramolecular peptide self-assembly.

The dynamic nature of supramolecular polymers has a key role in their organization. Yet, the manipulation of their dimensions and polarity remains a challenge. Here, the minimalistic diphenylalanine building block was applied to demonstrate control of nano-assemblies growth and shrinkage using microfluidics. To fine-tune differential local environments, peptide nanotubes were confined by micron-scale pillars and subjected to monomer flows of various saturation levels to control assembly and disassembly. The small-volume device allows the rapid adjustment of conditions within the system. A simplified kinetic model was applied to calculate parameters of the growth mechanism. Direct real-time microscopy analysis revealed that different peptide derivatives show unidirectional or bidirectional axial dimension variation. Atomistic simulations show that unidirectional growth is dictated by the differences in the axial ends, as observed in the crystalline order of symmetry. This work lays foundations for the rational control of nano-materials dimensions for applications in biomedicine and material science.