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We applied solid- and solution-state nuclear magnetic resonance spectroscopy to examine the structure of multidomain peptides composed of self-assembling ß-sheet domains linked to bioactive domains. Bioactive domains can be selected to stimulate specific biological responses (e.g., via receptor binding), while the ß-sheets provide the desirable nanoscale properties. Although previous work has established the efficacy of multidomain peptides, molecular-level characterization is lacking. The bioactive domains are intended to remain solvent-accessible without being incorporated into the ß-sheet structure. We tested for three possible anticipated molecular-level consequences of introducing bioactive domains to ß-sheet-forming peptides: (1) the bioactive domain has no effect on the self-assembling peptide structure; (2) the bioactive domain is incorporated into the ß-sheet nanofiber; and (3) the bioactive domain interferes with self-assembly such that nanofibers are not formed. The peptides involved in this study incorporated self-assembling domains based on the (SL)6 motif and bioactive domains including a VEGF-A mimic (QK), an IGF-mimic (IGF-1c), and a de novo SARS-CoV-2 binding peptide (SBP3). We observed all three of the anticipated outcomes from our examination of peptides, illustrating the unintended structural effects that could adversely affect the desired biofunctionality and biomaterial properties of the resulting peptide hydrogel. This work is the first attempt to evaluate the structural effects of incorporating bioactive domains into a set of peptides unified by a similar self-assembling peptide domain. These structural insights reveal unmet challenges in the design of highly tunable bioactive self-assembling peptide hydrogels.
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Nanofibras , Péptidos , Conformación Proteica en Lámina beta , Péptidos/química , Nanofibras/química , Hidrogeles/química , Materiales BiocompatiblesRESUMEN
Amyloid aggregates of specific proteins constitute important pathological hallmarks in many neurodegenerative diseases, defining neuronal degeneration and disease onset. Recently, increasing numbers of patients show comorbidities and overlaps between multiple neurodegenerative diseases, presenting distinct phenotypes. Such overlaps are often accompanied by colocalizations of more than one amyloid protein, prompting the question of whether direct interactions between different amyloid proteins could generate heterotypic amyloids. To answer this question, we investigated the effect of α-synuclein (αS) on the DNA-binding protein TDP-43 aggregation inspired by their coexistence in pathologies such as Lewy body dementia and limbic predominant age-related TDP-43 encephalopathy. We previously showed αS and prion-like C-terminal domain (PrLD) of TDP-43 synergistically interact to generate toxic heterotypic aggregates. Here, we extend these studies to investigate whether αS induces structurally and functionally distinct polymorphs of PrLD aggregates. Using αS-PrLD heterotypic aggregates generated in two different stoichiometric proportions, we show αS can affect PrLD fibril forms. PrLD fibrils show distinctive residue level signatures determined by solid state NMR, dye-binding capability, proteinase K (PK) stability, and thermal stability toward SDS denaturation. Furthremore, by gold nanoparticle labeling and transmission electron microscopy, we show the presence of both αS and PrLD proteins within the same fibrils, confirming the existence of heterotypic amyloid fibrils. We also observe αS and PrLD colocalize in the cytosol of neuroblastoma cells and show that the heterotypic PrLD fibrils selectively induce synaptic dysfunction in primary neurons. These findings establish the existence of heterotypic amyloid and provide a molecular basis for the observed overlap between synucleinopathies and TDP-43 proteinopathies.
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Nanopartículas del Metal , Enfermedades Neurodegenerativas , Síndromes de Neurotoxicidad , Humanos , alfa-Sinucleína/metabolismo , Oro , Amiloide/química , Enfermedades Neurodegenerativas/metabolismo , Proteínas de Unión al ADN/genéticaRESUMEN
The tunability of chromatic phases adapted by chromogenic polymers such as polydiacetylene (PDA) is key to their utility for robust sensing applications. Here, we investigated the influence of charged peptide interactions on the structure-dependent thermochromicity of amphiphilic PDAs. Solid-state NMR and circular dichroism analyses show that our oppositely charged peptide-PDA samples have distinct degrees of structural order, with the coassembled sample being in between the ß-sheet-like positive peptide-PDA and the relatively disordered negative peptide-PDA. All solutions exhibit thermochromicity between 20 and 80 °C, whereby the hysteresis of the blue, planar phase is much larger than that of the red, twisted phase. Resonance Raman spectroscopy of films demonstrates that only coassemblies with electrostatic complementarity stabilize coexisting blue and red PDA phases. This work reveals the nature of the structural changes responsible for the thermally responsive chromatic transitions of biomolecule-functionalized polymeric materials and how this process can be directed by sequence-dictated electrostatic interactions.
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Nanoestructuras , Poliinos , Poliinos/química , Polímero Poliacetilénico , Polímeros/química , PéptidosRESUMEN
Self-assembly of proteinaceous biomolecules into functional materials with ordered structures that span length scales is common in nature yet remains a challenge with designer peptides under ambient conditions. This report demonstrates how charged side-chain chemistry affects the hierarchical co-assembly of a family of charge-complementary ß-sheet-forming peptide pairs known as CATCH(X+/Y-) at physiologic pH and ionic strength in water. In a concentration-dependent manner, the CATCH(6K+) (Ac-KQKFKFKFKQK-Am) and CATCH(6D-) (Ac-DQDFDFDFDQD-Am) pair formed either ß-sheet-rich microspheres or ß-sheet-rich gels with a micron-scale plate-like morphology, which were not observed with other CATCH(X+/Y-) pairs. This hierarchical order was disrupted by replacing D with E, which increased fibril twisting. Replacing K with R, or mutating the N- and C-terminal amino acids in CATCH(6K+) and CATCH(6D-) to Qs, increased observed co-assembly kinetics, which also disrupted hierarchical order. Due to the ambient assembly conditions, active CATCH(6K+)-green fluorescent protein fusions could be incorporated into the ß-sheet plates and microspheres formed by the CATCH(6K+/6D-) pair, demonstrating the potential to endow functionality.
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Péptidos , Conformación Proteica en Lámina beta , Péptidos/química , GelesRESUMEN
Myocilin-associated glaucoma is a protein-conformational disorder associated with formation of a toxic amyloid-like aggregate. Numerous destabilizing single point variants, distributed across the myocilin olfactomedin ß-propeller (OLF, myocilin residues 245-504, 30 kDa) are associated with accelerated disease progression. In vitro, wild type (WT) OLF can be promoted to form thioflavin T (ThT)-positive fibrils under mildly destabilizing (37°C, pH 7.2) conditions. Consistent with the notion that only a small number of residues within a protein are responsible for amyloid formation, 3D 13C-13C solid-state NMR spectra show that OLF fibrils are likely to be composed of only about one third of the overall sequence. Here, we probe the residue composition of fibrils formed de novo from purified full-length OLF. We were able to make sequential assignments consistent with the sequence S331-G-S-L334. This sequence appears once within a previously identified amyloid-prone region (P1, G326AVVYSGSLYFQ) internal to OLF. Since nearly half of the pairs of adjacent residues (di-peptides) in OLF occur only once in the primary structure and almost all the 3-residue sequences (tri-peptides) are unique, remarkably few sequential assignments are necessary to uniquely identify specific regions of the amyloid core. This assignment approach could be applied to other systems to expand our molecular comprehension of how folded proteins undergo fibrillization.
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Bioinspired strategies have been given extensive attention for the recovery of rare earth elements (REEs) from waste streams because of their high selectivity, regeneration potential, and sustainability as well as low cost. Lanmodulin protein is an emerging biotechnology that is highly selective for REE binding. Mimicking lanmodulin with shorter peptides is advantageous because they are simpler and potentially easier to manipulate and optimize. Lanmodulin-derived peptides have been found to bind REEs, but their properties have not been explored when immobilized on solid substrates, which is required for many advanced separation technologies. Here, two peptides, LanM1 and scrambled LanM1, are designed from the EF-hand loop 1 of lanmodulin and investigated for their binding affinity toward different REEs when surface-bound. First, the ability of LanM1 to bind REEs was confirmed and characterized in solution using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations for Ce(III) ions. Isothermal titration calorimetry (ITC) was used to further analyze the binding of the LanM1 to Ce(III), Nd(III), Eu(III), and Y(III) ions and in low-pH conditions. The performance of the immobilized peptides on a model gold surface was examined using a quartz crystal microbalance with dissipation (QCM-D). The studies show that the LanM1 peptide has a stronger REE binding affinity than that of scrambled LanM1 when in solution and when immobilized on a gold surface. QCM-D data were fit to the Langmuir adsorption model to estimate the surface-bound dissociation constant (Kd) of LanM1 with Ce(III) and Nd(III). The results indicate that LanM1 peptides maintain a high affinity for REEs when immobilized, and surface-bound LanM1 has no affinity for potential competitor calcium and copper ions. The utility of surface-bound LanM1 peptides was further demonstrated by immobilizing them to gold nanoparticles (GNPs) and capturing REEs from solution in experiments utilizing an Arsenazo III-based colorimetric dye displacement assay and ultraviolet-visible (UV-vis) spectrophotometry. The saturated adsorption capacity of GNPs was estimated to be around 3.5 µmol REE/g for Ce(III), Nd(III), Eu(III), and Y(III) ions, with no binding of non-REE Ca(II) ions observed.
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Nanopartículas del Metal , Metales de Tierras Raras , Oro , Metales de Tierras Raras/química , Péptidos , IonesRESUMEN
Peptide self-assembly into amyloid fibrils provides numerous applications in drug delivery and biomedical engineering applications. We augment our previously-established computational screening technique along with experimental biophysical characterization to discover 7-mer peptides that self-assemble into "parallel ß-sheets", that is, ß-sheets with N-terminus-to-C-terminus ð½-strand vectors oriented in parallel. To accomplish the desired ß-strand organization, we applied the PepAD amino acid sequence design software to the Class-1 cross-ß spine defined by Sawaya et al. This molecular configuration includes two layers of parallel ß-sheets stacked such that N-terminus-to-C-terminus vectors are oriented antiparallel for molecules on adjacent ß-sheets. The first cohort of PepAD identified peptides were examined for their fibrillation behavior in DMD/PRIME20 simulations, and the top performing sequence was selected as a prototype for a subsequent round of sequence refinement. The two rounds of design resulted in a library of eight 7-mer peptides. In DMD/PRIME20 simulations, five of these peptides spontaneously formed fibril-like structures with a predominantly parallel ð½-sheet arrangement, two formed fibril-like structure with <50% in parallel ð½-sheet arrangement and one remained a random coil. Among the eight candidate peptides produced by PepAD and DMD/PRIME20, five were synthesized and purified. All five assembled into amyloid fibrils composed of parallel ß-sheets based on Fourier transform infrared spectroscopy, circular dichroism, electron microscopy, and thioflavin-T fluorescence spectroscopy measurements.
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Método de Montecarlo , Conformación Proteica en Lámina beta , Nanofibras/química , Péptidos/química , Secuencia de Aminoácidos , Estructura Secundaria de Proteína , Amiloide/química , Modelos Moleculares , Simulación de Dinámica MolecularRESUMEN
Q11 peptide nanofibers are used as a biomaterial for applications such as antigen presentation and tissue engineering, yet detailed knowledge of molecular-level structure has not been reported. The Q11 peptide sequence was designed using heuristics-based patterning of hydrophobic and polar amino acids with oppositely charged amino acids placed at opposite ends of the sequence to promote antiparallel ß-sheet formation. In this work, we employed solid-state nuclear magnetic resonance spectroscopy (NMR) to evaluate whether the molecular organization within Q11 self-assembled peptide nanofibers is consistent with the expectations of the peptide designers. We discovered that Q11 forms a distribution of molecular structures. NMR data from two-dimensional (2D) 13C-13C dipolar-assisted rotational resonance indicate that the K3 and E9 residues between Q11 ß-strands are spatially proximate (within â¼0.6 nm). Frequency-selective rotational echo double resonance (fsREDOR) on K3 Nζ and E9 Cδ-labeled sites showed that approximately 9% of the sites are close enough for salt bridge formation to occur. Surprisingly, dipolar recoupling measurements revealed that Q11 peptides do not assemble into antiparallel ß-sheets as expected, and structural analysis using Fourier-transform infrared spectroscopy and 2D NMR alone can be misleading. 13C PITHIRDS-CT dipolar recoupling measurements showed that the most abundant structure consists of parallel ß-sheets, in contrast to the expected antiparallel ß-sheet structure. Structural heterogeneity was detected from 15N{13C} REDOR measurements, with approximately 22% of ß-strands having antiparallel nearest neighbors. We cannot propose a complete structural model of Q11 nanofibers because of the complexity involved when examining structurally heterogeneous samples using NMR. Altogether, our results show that while heuristics-based patterning is effective in promoting ß-sheet formation, designing a peptide sequence to form a targeted ß-strand arrangement remains challenging.
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Nanofibras , Péptidos , Conformación Proteica en Lámina beta , Nanofibras/química , Péptidos/química , Resonancia Magnética Nuclear Biomolecular , Secuencia de AminoácidosRESUMEN
Growth factor (GF) mimicry involves recapitulating the signaling of larger molecules or cells. Although GF mimicry holds considerable promise in tissue engineering and drug design applications, difficulties in targeting the signaling molecule to the site of delivery and dissociation of mimicking peptides from their target receptors continue to limit its clinical application. To address these challenges, we utilized a self-assembling peptide (SAP) platform to generate synthetic insulin-like growth factor (IGF)-signaling, self-assembling GFs. Our peptide hydrogels are biocompatible and bind target IGF receptors in a dose-dependent fashion, activate proangiogenic signaling, and facilitate formation of angiogenic microtubules in vitro. Furthermore, infiltrated hydrogels are stable for weeks to months. We conclude that the enhanced targeting and long-term stability of our SAP/GF mimicry implants may improve the efficacy and safety of future GF mimic therapeutics.
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Péptidos Similares a la Insulina , Péptidos , Péptidos/química , Péptidos y Proteínas de Señalización Intercelular , Ingeniería de Tejidos , Hidrogeles/químicaRESUMEN
The lasting threat of viral pandemics necessitates the development of tailorable first-response antivirals with specific but adaptive architectures for treatment of novel viral infections. Here, such an antiviral platform has been developed based on a mixture of hetero-peptides self-assembled into functionalized ß-sheets capable of specific multivalent binding to viral protein complexes. One domain of each hetero-peptide is designed to specifically bind to certain viral proteins, while another domain self-assembles into fibrils with epitope binding characteristics determined by the types of peptides and their molar fractions. The self-assembled fibrils maintain enhanced binding to viral protein complexes and retain high resilience to viral mutations. This method is experimentally and computationally tested using short peptides that specifically bind to Spike proteins of SARS-CoV-2. This platform is efficacious, inexpensive, and stable with excellent tolerability.
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COVID-19 , Humanos , Péptidos/química , SARS-CoV-2/metabolismo , Antivirales/farmacología , Proteínas Virales , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Amyloid beta (Aß) aggregation is a slow process without seeding or assisted nucleation. Sodium dodecyl sulfate (SDS) micelles stabilize Aß42 small oligomers (in the dimer to tetramer range); subsequent SDS removal leads to a 150-kD Aß42 oligomer. Dodecylphosphorylcholine (DPC) micelles also stabilize an Aß42 tetramer. Here we investigate the detergent-assisted oligomerization pathway by solid-state NMR spectroscopy and molecular dynamics simulations. SDS- and DPC-induced oligomers have the same structure, implying a common oligomerization pathway. An antiparallel ß-sheet formed by the C-terminal region, the only stable structure in SDS and DPC micelles, is directly incorporated into the 150-kD oligomer. Three Gly residues (at positions 33, 37, and 38) create holes that are filled by the SDS and DPC hydrocarbon tails, thereby turning a potentially destabilizing feature into a stabilizing factor. These observations have implications for endogenous Aß aggregation at cellular interfaces.
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Péptidos beta-Amiloides , Detergentes , Péptidos beta-Amiloides/metabolismo , Micelas , Estructura Secundaria de ProteínaRESUMEN
Screening amino acid sequence space via experiments to discover peptides that self-assemble into amyloid fibrils is challenging. We have developed a computational peptide assembly design (PepAD) algorithm that enables the discovery of amyloid-forming peptides. Discontinuous molecular dynamics (DMD) simulation with the PRIME20 force field combined with the FoldAmyloid tool is used to examine the fibrilization kinetics of PepAD-generated peptides. PepAD screening of â¼10,000 7-mer peptides resulted in twelve top-scoring peptides with two distinct hydration properties. Our studies revealed that eight of the twelve in silico discovered peptides spontaneously form amyloid fibrils in the DMD simulations and that all eight have at least five residues that the FoldAmyloid tool classifies as being aggregation-prone. Based on these observations, we re-examined the PepAD-generated peptides in the sequence pool returned by PepAD and extracted five sequence patterns as well as associated sequence signatures for the 7-mer amyloid-forming peptides. Experimental results from Fourier transform infrared spectroscopy (FTIR), thioflavin T (ThT) fluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) indicate that all the peptides predicted to assemble in silico assemble into antiparallel ß-sheet nanofibers in a concentration-dependent manner. This is the first attempt to use a computational approach to search for amyloid-forming peptides based on customized settings. Our efforts facilitate the identification of ß-sheet-based self-assembling peptides, and contribute insights towards answering a fundamental scientific question: "What does it take, sequence-wise, for a peptide to self-assemble?".
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Myocilin-associated glaucoma is a new addition to the list of diseases linked to protein misfolding and amyloid formation. Single point variants of the â¼257-residue myocilin olfactomedin domain (mOLF) lead to mutant myocilin aggregation. Here, we analyze the 12-residue peptide P1 (GAVVYSGSLYFQ), corresponding to residues 326-337 of mOLF, previously shown to form amyloid fibrils in vitro and in silico. We applied solid-state NMR structural measurements to test the hypothesis that P1 fibrils adopt one of three predicted structures. Our data are consistent with a U-shaped fibril arrangement for P1, one that is related to the U-shape predicted previously in silico. Our data are also consistent with an antiparallel fibril arrangement, likely driven by terminal electrostatics. Our proposed structural model is reminiscent of fibrils formed by the Aß(1-40) Iowa mutant peptide, but with a different arrangement of molecular turn regions. Taken together, our results strengthen the connection between mOLF fibrils and the broader amylome and contribute to our understanding of the fundamental molecular interactions governing fibril architecture and stability.
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Glaucoma , Glicoproteínas , Amiloide , Péptidos beta-Amiloides , Proteínas del Citoesqueleto , Proteínas de la Matriz Extracelular , Proteínas del Ojo/genética , Glaucoma/genética , Glicoproteínas/genética , HumanosRESUMEN
Peptide coassembly, wherein at least two different peptides interact to form multicomponent nanostructures, is an attractive approach for generating functional biomaterials. Current efforts seek to design pairs of peptides, A and B, that form nanostructures (e.g., ß-sheets with ABABA-type ß-strand patterning) while resisting self-assembly (e.g., AAAAA-type or BBBBB-type ß-sheets). To confer coassembly behavior, most existing designs have been based on highly charged variants of known self-assembling peptides; like-charge repulsion limits self-assembly while opposite-charge attraction promotes coassembly. Recent analyses using solid-state NMR and coarse-grained simulations reveal that preconceived notions of structure and molecular organization are not always correct. This perspective highlights recent advances and key challenges to understanding and controlling peptide coassembly.