Sequence Decoding of 1D to 2D Self-Assembling Cyclic Peptides

This is the peer reviewed version of the following article: S. Díaz, I. Insua, G. Bhak, J. Montenegro, Chem. Eur. J. 2020, 26, 14765, which has been published in final form at https://doi.org/10.1002/chem.202003265. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.The inherent ability of peptides to self-assemble with directional and rationally predictable interactions has fostered a plethora of synthetic two-dimensional (2D) supramolecular biomaterials. However, the design of peptides with hierarchical assembly in different dimensions across mesoscopic lengths remains a challenging task. We here describe the structural exploration of a d/l-alternating cyclic octapeptide capable of assembling one-dimensional (1D) nanotubes in water, which subsequently pack laterally to form giant 2D nanosheets up to 500 μm long with a constant 3.2 nm thickness. Specific amino acid mutations allowed the mapping of structure–assembly relationships that determine 2D self-assembly. Nine peptide modifications were studied, revealing key features in the peptide sequence that nanosheets tolerated, while a total of three peptide variants included modifications that compromised their 2D arrangement. These lessons will serve as guide and inspiration for new 2D supramolecular peptide designsThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [SAF2017-89890-R], Xunta de Galicia (AD031 2016, ED431C 2017/25 and ED431G2019/03) and the European Commission (EC) (European Regional Development Fund-ERDF) Instituto de Salud Carlos III (COV20/00297). I.I. thanks the European Commission (EC) and AEI for MSCA-IF (2018-843332) and Juan de la Cierva (FJCI-2017-31795) fellowships, respectively. J.M. thanks the Ramón y Cajal (RYC-2013-13784), ERC- STG (DYNAP, 2016-677786), ERC-POC (TraffikGene, 2019-838002) and Human Frontier Science Programme Young Investigator Grant (RGY0066/2017) for fundingS

An Adhesive Peptide from the C-Terminal Domain of α-Synuclein for Single-Layer Adsorption of Nanoparticles onto Substrates

The two-dimensional (2D) homogeneous assembly of nanoparticle monolayer arrays onto a broad range of substrates constitutes an important challenge for chemistry, nanotechnology, and material science. α-Synuclein (αS) is an intrinsically disordered protein associated with neuronal protein complexes and has a high degree of structural plasticity and chaperone activity. The C-terminal domain of αS has been linked to the noncovalent interactions of this protein with biological targets and the activity of αS in presynaptic connections. Herein, we have systematically studied peptide fragments of the chaperone-active C-terminal sequence of αS and identified a 17-residue peptide that preserves the versatile binding nature of αS. Attachment of this short peptide to gold nanoparticles afforded colloidally stable nanoparticle suspensions that allowed the homogeneous 2D adhesion of the conjugates onto a wide variety of surfaces, including the formation of crystalline nanoparticle superlattices. The peptide sequence and the strategy reported here describe a new adhesive molecule for the controlled monolayer adhesion of metal nanoparticles and sets a stepping-stone toward the potential application of the adhesive properties of αSThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [BIO2015-70092-R, SAF2017-89890-R], the Xunta de Galicia (ED431C 2017/25, 2016-AD031, AGAUR (2017 SGR 324), and Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/09), the ISCIII (COV20/00297), and the European Union (European Regional Development Fund – ERDF). X.S. acknowledges funding from the ERC (CONCERT-648201). IRB Barcelona is the recipient of a Severo Ochoa Award (Government of Spain). J.M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Grant (DYNAP-677786), and a Young Investigator Grant from the HFSP (RGY0066/2017)S

Monitoring the Formation of Amyloid Oligomers Using Photoluminescence Anisotropy

The formation of oligomeric soluble aggregates is related to the toxicity of amyloid peptides and proteins. In this manuscript, we report the use of a ruthenium polypyridyl complex ([Ru(bpy)2(dpqp)]2+) to track the formation of amyloid oligomers at different times using photoluminescence anisotropy. This technique is sensitive to the rotational correlation time of the molecule under study, which is consequently related to the size of the molecule. [Ru(bpy)2(dpqp)]2+ presents anisotropy values of zero when free in solution (due to its rapid rotation and long lifetime) but larger values as the size and concentration of amyloid-β (Aβ) oligomers increase. Our assays show that Aβ forms oligomers immediately after the assay is started, reaching a steady state at ∼48 h. SDS–PAGE, DLS, and TEM were used to confirm and characterize the formation of oligomers. Our experiments show that the rate of formation for Aβ oligomers is temperature dependent, with faster rates as the temperature of the assay is increased. The probe was also effective in monitoring the formation of α-synuclein oligomers at different timesAAM thanks the Welch Foundation (Grant C-1743) and JM thanks AEI (SAF2017-89890-R), ERC (DYNAP-677786) and HFSP (RGY0066/2017) for financial supportS

Granular Assembly of α-Synuclein Leading to the Accelerated Amyloid Fibril Formation with Shear Stress

α-Synuclein participates in the Lewy body formation of Parkinson's disease. Elucidation of the underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop a controlling strategy toward the disease, but also to apply the protein fibrils for future biotechnology. Discernable homogeneous granules of α-synuclein composed of approximately 11 monomers in average were isolated in the middle of a lag phase during the in vitro fibrillation process. They were demonstrated to experience almost instantaneous fibrillation during a single 12-min centrifugal membrane-filtration at 14,000×g. The granular assembly leading to the drastically accelerated fibril formation was demonstrated to be a result of the physical influence of shear force imposed on the preformed granular structures by either centrifugal filtration or rheometer. Structural rearrangement of the preformed oligomomeric structures is attributable for the suprastructure formation in which the granules act as a growing unit for the fibril formation. To parallel the prevailing notion of nucleation-dependent amyloidosis, we propose a double-concerted fibrillation model as one of the mechanisms to explain the in vitro fibrillation of α-synuclein, in which two consecutive concerted associations of monomers and subsequent oligomeric granular species are responsible for the eventual amyloid fibril formation

Self-Assembled Micro-Fibres by Oxime Connection of Linear Peptide Amphiphiles

Linear peptide amphiphiles are excellent biocompatible scaffolds for the hierarchical self-assembly of one dimensional nanostructures in aqueous media. However, their structural exploration and screening of self-assembling properties is often limited by time-consuming synthesis and purification steps. We here describe the application of the oxime bond as a powerful synthetic tool towards the conjugation of peptide heads bearing a hydroxylamine group with hydrophobic aldehyde tails. This methodology allowed the quick prepraration of a small library of oxime-connected peptide amphiphiles, whose supramolecular screening revealed nano-to-micro fibrillation with dependency on their chemical structure. These results demonstrate the simplicity and the synthetic potential of the oxime conjugation for the preparation of peptide amphiphiles with improved self-assembling capabilitiesThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [CTQ2014-59646-R, SAF2017-89890-R], the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. I.I. thanks the Spanish AEI for a Juan de la Cierva -Formación fellowship (FJCI-2017-31795). J.M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S

In Situ Fibril Formation of κ‑Casein by External Stimuli within Multilayer Thin Films

We have developed the in situ fibrillation of κ-casein, employed as amyloid precursor, within multilayer films consisting of κ-casein and poly­(acrylic acid) (PAA) prepared by the layer-by-layer (LbL) deposition. The fibrillation of κ-casein within the multilayered films is strongly dependent on the extent of intermolecular interactions between κ-casein and PAA. When films constructed initially at pH 3 were heat treated at the same pH, κ-casein did not transform into fibrils. However, when the films were subjected to heat treatment at pH 5, κ-casein was transformed into fibrils within multilayer films due to weakened intermolecular interactions between κ-casein and PAA. We also noted that the multilayer film was swollen at pH 5 by the charge imbalance within the film, which we believe gives enough mobility for κ-caseins to form fibrils with adjacent κ-caseins within the multilayer. The fibrils were found to be uniformly distributed across the entire film thickness, and the aspect ratio as well as the number density of fibrils increased as a function of incubation time. The present study reveals a strategy to realize in situ nanocomposites within LbL multilayer films simply by triggering the formation of protein fibrils by controlling the intermolecular interactions between amyloid precursors and polyelectrolytes (PEs)

Instantaneous Amyloid Fibril Formation of α-Synuclein from the Oligomeric Granular Structures in the Presence of Hexane

Amyloid fibrils found in various neurodegenerative disorders are also recognized as high-performance protein nanomaterials with a formidable rigidity. Elucidation of an underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop controlling strategy toward the diseases, but also to apply the protein fibrils for future nanobiotechnology. α-Synuclein is an amyloidogenic protein responsible for the radiating filament formation within Lewy bodies of Parkinson's disease. The amyloid fibril formation of α-synuclein has been shown to be induced from the oligomeric granular species of the protein acting as a growing unit by experiencing structural rearrangement within the preformed oligomeric structures in the presence of an organic solvent of hexane. This granule-based concerted amyloid fibril formation model would parallel the prevalent notion of nucleation-dependent fibrillation mechanism in the area of amyloidosis