Zein, a protein found in maize endosperm is a major co-product of the bio-fuel industry with different application in (i) biomedicine as drug-delivery compound, drug capsules that dissolve in the body; (ii) in industrial production of bioplastic, paper coating, and food products (chewing gum). Zein was classified according to the structure of the amino acid sequence, solubilization and molecular weight into 4 fractions, α, β, γ and δ-zeins. The α-zein is found in the largest amount and consists in two peptides with 19 kDa and 22 kDa and is the extraction is usually perform with ethanol. Due to the high complexity of the starting material and to the extraction conditions, analysis of protein compositions requires a combination of modern analytical methods such as SDS-PAGE, 2D gel electrophoresis and mass spectrometry. Here, we investigate, by using analytical methods, the efficiency of zein extraction from corn flours using 65-95% aqueous ethanol under ultrasound conditions from dry-ground whole corn as well as meals with different grain sizes. Moreover, the extracted zein and the commercial zein protein were used for different conjugates synthesis. We have applied modern methods such as MALDI ToF mass spectrometry, SDS-PAGE electrophoresis and FT-IR spectroscopy to characterize the extracted zein.

Acknowledgments: „This work was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI – UEFISCDI, project number PN-III-P1-1.1-PD-2019-0442, within PNCDI III”.

Hierarchical phosphorylation of the disordered 4E-BP2 protein stabilizes a binding incompatible 4-stranded beta domain while the C-terminal domain remains disordered. Ensemble descriptions of both phosphorylation states were calculated. The ensembles were restrained using Small-angle X-ray scattering (SAXS) and Paramagnetic Resonance Enhancement (PRE), while the single-molecule Förster Resonance Energy Transfer (smFRET) between residues 32 and 91 was used as validation of the ability of the restrained ensemble to agree with independent experimental evidence.

Initially, conformational ensembles were calculated using ENSEMBLE¹ and SAXS-only restraints on an initial pool of 4E-BP2 conformers generated in TraDES.² Bimodal distributions of the radius of gyration (R_G) were obtained for both phospho forms of the protein. For non-phospho 4E-BP2, the back-calculated FRET efficiency was lower than the experimental value, while for the five-phospho 4E-BP2, the opposite happened. Adding PRE restraints for the five-phospho form, amplified the disagreement with the smFRET data. For both phospho forms, the average hydrodynamic radius (R_H) of the calculated ensemble was smaller than the experimental value determined by Fluorescence Correlation Spectroscopy (FCS). These discrepancies highlight the inability of the TraDES prior to capture the secondary structure of 4E-BP2. Alternatively, we used a new Rosetta-based method (Fast Floppy Tail, FFT)³ to generate initial pools of conformations for the two 4E-BP2 phosphoforms. Applying SAXS, PRE, chemical shifts and hydrodynamic restraints in ENSEMBLE on these FFT-generated initial pools lead to better agreement with smFRET data and to a more accurate ensemble representation of 4E-BP2 in its two functionally-relevant forms.

1. Feldman, H. J., & Hogue, C. W. (2000). Proteins: Structure, Function, and Bioinformatics, 39, 112-131.
2. Krzeminski, Mickaël, et al. Bioinformatics29.3 (2013): 398-399.
3. Ferrie, J. J., & Petersson, E. J. (2020). The Journal of Physical Chemistry B.

SARS-COV-2 has evolved into a pandemic of unprecedent scale. This coronavirus enters cells by the interaction of the Receptor Binding Domain (RBD) with the human Angiotensin-Converting Enzyme 2 receptor (hACE-2). In this study, we employed a rational structure-based design to propose 22-mer stapled peptides using the structure of the hACE2 α-1 helix as template. These peptides were designed to retain the alfa-helical character of the natural structure, to enhance binding affinity and to display a better solubility profile when compared to other designed peptides available in the literature. We employed different docking strategies (PATCHDOCK and ZDOCK) followed by a double-step refinement process (FIBERDOCK) to rank our peptides, followed by stability analysis/evaluation of the interaction profile of the best docking predictions using a 500 ns Molecular Dynamics (MD) simulation, and a further binding affinity analysis by the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method. Our best structure presented a stable profile and could retain important interactions with the RBD even in the presence of the E484K RBD mutation. We predict this peptide can bind the viral RBD with similar potency to the control NYBSP-4 (a 30-mer experimentally proven peptide inhibitor) displaying the advantages of being a smaller peptide. Furthermore, our study provides valuable information for the rational design of double-stapled peptide as inhibitors of SARS-CoV-2 infection.

Fluorescent probes and techniques can be used to study protein structures. Molecular processes that occur in living organisms are visualized through the use of fluorescence techniques, which are sensitive enough to detect miniscule changes. The intracellular details of G protein-coupled receptors (GPCRs) tagged with fluorescent dyes can be visualized by using fluorescence imaging microscopy. Confocal microscopy can detect sub-cellular structures, and by combining Forster resonance energy transfer (FRET) techniques with fluorescence microscopy, one can determine physical molecular interactions within proteins. By using fluorescence spectroscopy, spatial information regarding the molecular behaviour of fluorescent molecules can be determined by using a variety of fluorescence parameters: quantum yield, which relates to intensity, spectrum, lifetime, and anisotropy. These techniques are extremely sensitive to changes and minimally invasive. FRET techniques were applied to characterize and detail the conformational changes exhibited by the A2A Adenosine receptor reconstituted in HDL nanodiscs, labelled with Alexa 488 and Alexa 647 dyes at the T119C and Q226C positions on transmembrane helices 4 and 6. A variety of ligand conditions would induce unique separation distances between these helices, resulting in FRET efficiency values which correspond to distinct conformations of the receptor. Specifically, single-molecule Forster resonance energy transfer (smFRET) was performed on the receptor to distinguish active and inactive states, pertaining to different FRET efficiency values. Characterization of the sample involved using many fluorescence techniques, such as fluorescence correlation spectroscopy (FCS), emission and excitation spectra, fluorescence anisotropy decay (FAD), and fluorescence lifetime.

Self-assembled peptide-based hydrogels have shown promising properties for various applications, including resonance imaging and drug delivery. Here, the hydrogels’ molecules self-assemble into intertwined fibrillar structures through the cooperative effect of different non-covalent intra- and intermolecular interactions. Particularly, the peptide-based hydrogels are highly advantageous as nanocarriers for antitumour drug delivery owing to the low critical gelation concentration, amenable synthesis and structural tailoring of the rheological properties, as well as the biocompatibility and similarity to the extracellular matrix.

Lately, wider developments in the use of peptide-based supramolecular hydrogels with optimum drug delivery and mechanical properties in biomedical research and clinical translation are hampered by the limited commercial availability and prohibitive costs.

Hereby, considering the structural aspects that influence the assembly of peptide-based hydrogelators, a library of Cbz-protected dehydrodipeptides was synthesised and evaluated as minimalist hydrogels. The molecular aggregation, self-assembly, gelation and biocompatibility were thoroughly studied through fluorescence spectroscopy, and the mechanical properties were assessed. The compounds that afforded hydrogels were evaluated as drug delivery systems for curcumin and doxorubicin using biomembrane models. The hydrogels displayed similar properties to more complex supramolecular hydrogels. Moreover, the expeditious and scalable synthesis using amenable reaction conditions makes the Cbz-protected dehydrodipeptide hydrogels available at affordable cost to the research community.

Interleukin-1β (IL-1β) is a central pro-inflammatory cytokine. On binding to its receptor (IL-1R), IL-1β plays roles in labor, inflammation and immune response against invading pathogens. Premature birth occurs in about 10% of all births worldwide and may lead to morbidity and long-term health problems. The inflammatory component of premature birth may be harmful to the newborn. Although current therapeutic interventions may delay birth, they have no effect on inflammation. Our presentation will focus on the IL-1R modulating peptide 101.10 (H-D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH₂), which delays labor and curbs inflammation without effect on immune vigilance. Employing lactams analogues of 101.10, information has been obtained regarding the active conformation. Moreover, lead lactam analogs offer promise for delaying labor and improving neonatal outcomes [1-2].

References

1. Geranurimi, A.; Cheng, C. W.; Quiniou, C.; Zhu, T.; Hou, X.; Rivera, J. C.; St-Cyr, D. J.; Beauregard, K.; Bernard-Gauthier, V.; Chemtob, S. Probing Anti-inflammatory Properties Independent of NF-κB Through Conformational Constraint of Peptide-bases Interleukin-1β Receptor Biased Ligand. Front. Chem. 2019, 7, 23.

2. Geranurimi, A.; Cheng, C. W. H.; Quiniou, C.; Côté, F.; Hou, X.; Lahaie, I.; Boudreault, A.; Chemtob, S.; Lubell, W. D. Interleukin-1 Receptor Modulation Using β-Substituted α-Amino-γ-Lactam Peptides From Solid-Phase Synthesis and Diversification. Front. Chem. 2020, 8 (1182).

Since the approval of the first peptide therapeutic agent, the 51-amino acid (AA) hormone insulin, in 1923, drug discovery has progressively expanded into the chemical space between small molecules and large proteins. Subsequently, a significant number of peptides (and peptidomimetics) have received regulatory approval, reaching over 100 approved and marketed peptides in 2020. However, unlike small-molecule drugs, there is no general set of guidelines for designing a successful peptide-based drug and this makes the peptide chemists’ job of designing future peptide drug candidates challenging.

We have performed a detailed structural analysis on the approved peptide therapeutics and diagnostics, providing an overview of their key compositional trends to help guide the design of future peptide medicines. In detail, molar mass distribution, amino acid frequency, peptide modifications, macrocycles, N- and C-termini, origin of peptide design, and polar/lipophilic contribution of each member present in a given approved peptide have been investigated.

Over a total of 105 peptide pharmaceuticals analysed, 86% are natural or naturally-derived. Moreover, a bimodal distribution of peptide molar mass has emerged, with the large majority < 2000 g/mol. Among all the peptide constitutional members, most (around 81%) are represented by natural L-amino acids, while the residual 19% comprises non-natural AAs and modifications. A balance between polar and hydrophobic residues have been found within the peptide structures. Finally, 46% of the approved peptides are cyclic and 5 to 7 members cycles are the most common.

The data collected have been made freely available on PepTherDia (http://peptherdia.herokuapp.com), an online open-source database. Forecasting a starring role for peptides in the coming decades, we anticipate that new clear trends and, perhaps, rules in structural composition will emerge beyond our observations, leading to improved rational peptide drug design.

Hydrogen bonding plays a critical role in the self-assembly of peptide amphiphiles (PAs). Herein, we studied how to tune the macromolecular nanostructures’ property by manipulating the PA’s chemical structure with urea, a hydrogen bond donor. We designed and synthesized three PAs: urea modified PA, original PA, and longer hydrophobic tail PA with peptide sequence FFEE. The effect of pH, temperature, self-assembly pathway was studied by Transition Electron Microscopy, Atomic Force Microscopy, Circular Dichroism, and Small-Angle X-ray Scattering. From these studies, we found that the urea motif can change PA’s morphology and secondary structure at neutral pH while enhancing the physical stability against pH and temperature changes. The resulted hydrogel of urea PA showed better ability in the shear recovery test than the others. Computational modeling revealed the packing and mechanism of PA-assembled nanostructures at the atomic level, clearly showing that there are greater numbers of hydrogen bonds for the PA with urea motif than for the others by a factor of 1.4. Furthermore, the urea-phenyl interaction makes the peptide portion more compact than it is in the absence of urea. This study helps to clarify the mechanism of hydrogen bonding acting on PA’s nanostructures with the presence of π-π stacking and will thereby help in the design of a new generation of PAs.

Perylenediimide derivatives (PDIs) constitute an important group of an organic π-conjugated system that has been abundantly studied owing to their excellent photostability, chemical robustness, synthetic accessibility, and optoelectronic properties. Assembling PDI molecules into tailored supramolecular structures leads to unique photochemical functionalities that can be exploited in various fields, from biomedicine to material sciences. However, the precise control over the directionality of PDI self-assembly into defined nanostructures remains problematic. In this study, we took advantage of the high aggregation propensity of amyloid peptides to design nanofilaments functionalized with PDI. The amyloidogenic domain of the islet polypeptide (IAPP), i.e., the segment 20-29 (I_20-29), was used as the self-assembling core, which was connected to PDI by a flexible hexyl spacer. PDI-[Leu]₂ derivative was prepared from perylene tetracarboxylic dianhydride (PTCDA), before being conjugated on the N-terminal amine of the elongated protected peptidyl-resin. By modulating the stoichiometry of the coupling reaction, two types of hybrid peptides were obtained: symmetric (PDI-[I_20-29]₂) and asymmetric (PDI-I_20-29). Self-assembly was initiated by the successive dispersion of PDI-[I_20-29]₂ and PDI-I_20-29 in an aqueous buffer and the solution was constantly rotary agitated at room temperature. The self-assembly process was periodically followed by measuring absorbance and fluorescence of PDI, by circular dichroism (CD) spectroscopy, and by atomic force microscopy. The analyses revealed that both PDI-peptides self-assembled into long, unbranched, and linear filaments with a cross-β-sheet quaternary organization. The UV-vis absorbance spectra exhibit a clear signature of H-aggregates for both assemblies. Overall, this study exposes that defined nanostructures functionalized with PDI can be obtained from amyloid peptide building blocks, opening to novel applications in bioimaging, photodynamic therapy, and bioelectronic.

Cathelicidins are one of the major classes of host defense peptides in vertebrates. These peptides display broad spectrum anti-infective properties against several pathogens and are also able to modulate host immune responses which makes them attractive candidates for novel therapeutics.

A bioinformatics approach was used to identify novel cathelicidin sequences from the genomes of four crocodylian species. Based on certain physico-chemical parameters (such as charge, helicity and amphipathicity) combined with phylogenetic analysis, four cathelicidin peptides were selected for chemical synthesis and characterization. The antimicrobial and antibiofilm activity of the peptides was assessed in vitro and in vivo against several bacterial pathogens of medical importance using an organoid model of skin biofilm infection and a murine abscess model.

The crocodylian cathelicidins showed broad and potent in vitro antimicrobial and antibiofilm activity against several species of bacteria. Notably, As-CATH8 and Gg-CATH5 showed good eradication capacity against Staphyloccocus aureus biofilms in an organoid model of biofilm infected skin, outperforming the human cathelicidin LL-37. As-CATH8 was also superior to LL-37 against bacterial abscesses arising from S. aureus and Acinetobacter baumannii in a murine model of high density bacterial infection. In this case, treatment with As-CATH8 significantly decreased the number of bacteria recovered from the abscess after three days and also reduced the abscess size. Overall, this work highlights the therapeutic potential of crocodylian cathelicidins against bacterial infections.