Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.
Nucleic acids have been among the first targets for antitumor drugs and antibiotics, and with the unveiling of new biological roles in regulation of gene expression, specific DNA and RNA structures have become very attractive targets, especially when the corresponding proteins are undruggable. Biophysical assays to test target structure and ligand binding stoichiometry, affinity, specificity and binding modes are part of the drug development pipeline. Mass spectrometry offers unique advantages as a biophysical method due to its ability to distinguish each stoichiometry present in a mixture. In addition, advanced mass spectrometry approaches (reactive probing, fragmentation techniques, ion mobility spectrometry, ion spectroscopy) provide more detailed information on the complexes. Here we review the fundamentals of mass spectrometry and all its particularities when studying non-covalent nucleic acid structures, and then review what has been learned thanks to mass spectrometry on nucleic acid structures, self-assemblies (e.g., duplexes or G-quadruplexes), and their complexes with ligands. 47
The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N- and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections.
Recently a state-specific multireference perturbation theory (SSMRPT) with an improved virtual orbitals complete active space configuration interaction (IVO-CASCI) reference function has been proposed for treating electronic structures of radicals such as methylene, m-benzyne, pyridyne, and pyridynium cation. This new development in MRPT, termed as IVO-SSMRPT, ensures that it is able to describe the structure of radicaloids with reasonable accuracy even with small reference spaces. IVO-SSMRPT is also capable of predicting the correct ordering of the lowest singlet-triplet gaps. Investigation of the first three electronic states of the oxygen molecule has also been used for rating our method. The agreement of our estimates with the available far more expensive benchmark state-of-the-art ab initio calculations is creditable. The IVO-SSMRPT method provides an effective avenue with manageable cost/accuracy ratio for accurately dealing with radicaloid systems possessing varying degrees of quasidegeneracy.
Molecular self-assembly, a phenomenon widely observed in nature, has been exploited through organic molecules, proteins, DNA and peptides to study complex biological systems. These self-assembly systems may also be used in understanding the molecular and structural biology which can inspire the design and synthesis of increasingly complex biomaterials. Specifically, use of these building blocks to investigate protein folding and misfolding has been of particular value since it can provide tremendous insights into peptide aggregation related to a variety of protein misfolding diseases, or amyloid diseases (e.g. Alzheimer’s disease, Parkinson’s disease, type-II diabetes). Herein, the self-assembly of TK9, a 9 residue peptide of the extra membrane C-terminal tail of the SARS Corona virus envelope, and its variants were characterized through biophysical, spectroscopic and simulated studies, and it was confirmed that the structure of these peptides influence their aggregation propensity, hence, mimicking amyloid proteins. TK9, which forms a beta-sheet rich fibril, contains a key sequence motif that may be critical for beta-sheet formation, thus making it an interesting system to study amyloid fibrillation. TK9 aggregates were further examined through simulations to evaluate the possible intra- and inter peptide interactions at the molecular level. These self-assembly peptides can also serve as amyloid inhibitors through hydrophobic and electrophilic recognition interactions. Our results show that TK9 inhibits the fibrillation of hIAPP, a 37 amino acid peptide implicated in the pathology of type-II diabetes. Thus, biophysical and NMR experimental results have revealed a molecular level understanding of peptide folding events, as well as the inhibition of amyloid-protein aggregation are reported.
Altered intestinal
permeability has been correlated with Parkinson’s
pathophysiology in the enteric nervous system, before manifestations
in the central nervous system (CNS). The inflammatory endotoxin or
lipopolysaccharide (LPS) released by gut bacteria is known to modulate
α-synuclein amyloidogenesis through the formation of intermediate
nucleating species. Here, biophysical techniques in conjunction with
microscopic images revealed the molecular interaction between lipopolysaccharide
and α-synuclein that induce rapid nucleation events. This heteromolecular
interaction stabilizes the α-helical intermediates in the α-synuclein
aggregation pathway. Multitude NMR studies probed the residues involved
in the LPS-binding structural motif that modulates the nucleating
forms, affecting the cellular internalization and associated cytotoxicity.
Collectively, our data characterizes this heteromolecular interaction
associated with an alternative pathway in Parkinson’s disease
progression.
Using NMR to probe transient binding of Aβ1–40 monomers to fibers, we find partially bound conformations with the highest degree of interaction near F19–K28 and a lesser degree of interaction near the C-terminus (L34–G37).
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