Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles

The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.

Immuno-Informatics Analysis of Pakistan-Based HCV Subtype-3a for Chimeric Polypeptide Vaccine Design

Hepatitis C virus (HCV) causes chronic and acute hepatitis infections. As there is extreme variability in the HCV genome, no approved HCV vaccine has been available so far. An effective polypeptide vaccine based on the functionally conserved epitopes will be greatly helpful in curing disease. For this purpose, an immuno-informatics study is performed based on the published HCV subtype-3a from Pakistan. First, the virus genome was translated to a polyprotein followed by a subsequent prediction of T-cell epitopes. Non-allergenic, IFN-γ producer, and antigenic epitopes were shortlisted, including 5 HTL epitopes and 4 CTL, which were linked to the final vaccine by GPGPG and AAY linkers, respectively. Beta defensin was included as an adjuvant through the EAAAK linker to improve the immunogenicity of the polypeptide. To ensure its safety and immunogenicity profile, antigenicity, allergenicity, and various physiochemical attributes of the polypeptide were evaluated. Molecular docking was conducted between TLR4 and vaccine to evaluate the binding affinity and molecular interactions. For stability assessment and binding of the vaccine-TLR4 docked complex, molecular dynamics (MD) simulation and MMGBSA binding free-energy analyses were conducted. Finally, the candidate vaccine was cloned in silico to ensure its effectiveness. The current vaccine requires future experimental confirmation to validate its effectiveness. The vaccine construct produced might be useful in providing immune protection against HCV-related infections.

Engineering and characterization of a novel Self Assembling Protein for Toxoplasma peptide vaccine in HLA-A*11:01, HLA-A*02:01 and HLA-B*07:02 transgenic mice

Fighting smart diseases requires smart vaccines. Novel ways to present protective immunogenic peptide epitopes to human immune systems are needed. Herein, we focus on Self Assembling Protein Nanoparticles (SAPNs) as scaffolds/platforms for vaccine delivery that produce strong immune responses against Toxoplasma gondii in HLA supermotif, transgenic mice. Herein, we present a useful platform to present peptides that elicit CD4+, CD8+ T and B cell immune responses in a core architecture, formed by flagellin, administered in combination with TLR4 ligand-emulsion (GLA-SE) adjuvant. We demonstrate protection of HLA-A*11:01, HLA-A*02:01, and HLA-B*07:02 mice against toxoplasmosis by (i) this novel chimeric polypeptide, containing epitopes that elicit CD8+ T cells, CD4+ T helper cells, and IgG2b antibodies, and (ii) adjuvant activation of innate immune TLR4 and TLR5 pathways. HLA-A*11:01, HLA-A*02:01, and HLA-B*07:02q11 transgenic mouse splenocytes with peptides demonstrated predicted genetic restrictions. This creates a new paradigm-shifting vaccine approach to prevent toxoplasmosis, extendable to other diseases.

An alpha/beta chimeric peptide molecular brush for eradicating MRSA biofilms and persister cells to mitigate antimicrobial resistance

An α/β chimeric polypeptide molecular brush shows excellent performance in eradicating established biofilms, persister cells, and clinically isolated multi-drug resistant Gram-positive bacteria.

Fibrillar Self-Assembly of a Chimeric Elastin-Resilin Inspired Engineered Polypeptide

In the field of tissue engineering, recombinant protein-based biomaterials made up of block polypeptides with tunable properties arising from the functionalities of the individual domains are appealing candidates for the construction of medical devices. In this work, we focused our attention on the preparation and structural characterization of nanofibers from a chimeric-polypeptide-containing resilin and elastin domain, designed on purpose to enhance its cell-binding ability by introducing a specific fibronectin-derived Arg-Gly-Asp (RGD) sequence. The polypeptide ability to self-assemble was investigated. The molecular and supramolecular structure was characterized by Scanning Electronic Microscopy (SEM) and Atomic Force Microscopy (AFM), circular dichroism, state-of-the-art synchrotron radiation-induced techniques X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The attained complementary results allow us to assess as H-bonds influence the morphology of the aggregates obtained after the self-assembling of the chimeric polypeptide. Finally, a preliminary investigation of the potential cytotoxicity of the polypeptide was performed by culturing human fetal foreskin fibroblast (HFFF2) for its use as biomedical device.

Искусственные электрон-транспортные цепи на основе зеленого флуоресцентного белка-=SUP=-*-=/SUP=-

In this paper, we combined a photoinduced electron donor—an improved green fluorescent protein (EGFP)—and protein oxidants within the same chimeric polypeptide chain. Comparison of the photostability of EGFP and chimeric proteins both in the absence and in the presence of non-protein-bound oxidants in solution showed the efficiency of the created model electron transport chains.

Insights into the effect of the J-domain on the substrate binding domain (SBD) of the 70 kDa heat shock protein, Hsp70, from a chimeric human J-SBD polypeptide

DnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain, a crucial event for the function that this system has in assisting protein folding. The interaction between Hsp40 and Hsp70 is transient and thus difficult to study, since mixing the binding partners can lead to quick dissociation due to their low affinity, creating a challenge for detailed analysis. As a consequence, knowledge of many important aspects of the mechanism of interaction is still lacking, for instance, the effect that J-domain binding has on Hsp70. In this study, we investigated whether it would be possible to gain understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale for this is that an increase in the proximity between the interacting partners in this engineered chimera will promote the natural interaction and facilitate the characterization of the protein– protein interaction, which is a requirement to gain further understanding of many biological processes. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the SBD alone. J-SBD behaved primarily as a monomer in all conditions tested and exhibited chaperone activity, as shown by aggregation protection and substrate binding assays, which revealed decreased binding to bis-ANS, a probe for hydrophobic patches. Collectively, our results suggest that Hsp40 binding to Hsp70 via the J-domain shifts the Hsp70 equilibrium towards the monomer state to expose hydrophobic sites prone to substrate accommodation.AbbreviationsBis-ANS (4,4’-Dianilino-1,1’-Binaphthyl-5,5’-Disulfonic Acid; CD, circular dichroism; Hsp, heat shock protein; J-SBD, chimeric polypeptide in which the J-domain of Hsp40 (at the N-terminus) is covalently attached to the substrate binding domain of Hsp70 (at the C-terminus) by a flexible linker; SBD: substrate binding domain of Hsp70.