Effect of Solvent on the Self-Assembly of Dialanine and Diphenylalanine Peptides

Diphenylalanine (FF) is a very common peptide with many potential applications, both biological and technological, due to a large number of different nanostructures which it attains. The current work concerns a detailed study of the self assembled structures of FF in two different solvents, an aqueous (H2O) and an organic (CH3OH) through simulations and experiments. Detailed atomistic Molecular Dynamics (MD) simulations of FF in both solvents have been performed, using an explicit solvent model. The self assembling propensity of FF in water is obvious while in methanol a very weak self assembling propensity is observed. We studied and compared structural properties of FF in the two different solvents and a comparison with a system of dialanine (AA) in the corresponding solvents was also performed. In addition, temperature dependence studies were carried out. Finally, the simulation predictions were compared to new experimental data, which were produced in the framework of the present work. A very good qualitative agreement between simulation and experimental observations was found

Designer TGFβ Superfamily Ligands with Diversified Functionality

Transforming Growth Factor – beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values

Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required

Self-Assembled Proteins and Peptides as Scaffolds for Tissue Regeneration

Self-assembling proteins and peptides are increasingly gaining interest for potential use as scaffolds in tissue engineering applications. They self-organize from basic building blocks under mild conditions into supramolecular structures, mimicking the native extracellular matrix. Their properties can be easily tuned through changes at the sequence level. Moreover, they can be produced in sufficient quantities with chemical synthesis or recombinant technologies to allow them to address homogeneity and standardization issues required for applications. Here. recent advances in self-assembling proteins, peptides, and peptide amphiphiles that form scaffolds suitable for tissue engineering are reviewed. The focus is on a variety of motifs, ranging from minimalistic dipeptides, simplistic ultrashort aliphatic peptides, and peptide amphiphiles to large "recombinamer" proteins. Special emphasis is placed on the rational design of self-assembling motifs and biofunctionalization strategies to influence cell behavior and modulate scaffold stability. Perspectives for combination of these "bottom-up" designer strategies with traditional "top-down" biofabrication techniques for new generations of tissue engineering scaffolds are highlighted. Recent advances in self-assembling proteins, peptides, and peptide amphiphiles that form scaffolds suitable for tissue engineering are discussed. Rational design and biofunctionalization strategies for a variety of motifs ranging from minimalistic dipeptides, ultrashort aliphatic peptides, and peptide amphiphiles to large "recombinamer" proteins are reviewed and challenges and perspectives for their widespread adoption in applications are highlighted. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Bacterial inclusion bodies are industrially exploitable amyloids

Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology.info:eu-repo/semantics/acceptedVersio

Bacterial inclusion bodies are industrially exploitable amyloids

Altres ajuts: CERCA Programme/Generalitat de CatalunyaUnderstanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology

The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis

Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse® were well correlated with sonication. Two other methods, Bugbuster® and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability

Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution

Identification and Localization of Proteins Associated with Biomineralization in the Iron Deposition Vesicles of Honeybees (Apis mellifera)

Honeybees (Apis mellifera) form superparamagnetic magnetite to act as a magnetoreceptor for magnetoreception. Biomineralization of superparamagnetic magnetite occurs in the iron deposition vesicles of trophocytes. Even though magnetite has been demonstrated, the mechanism of magnetite biomineralization is unknown. In this study, proteins in the iron granules and iron deposition vesicles of trophocytes were purified and identified by mass spectrometry. Antibodies against such proteins were produced. The major proteins include actin, myosin, ferritin 2, and ATP synthase. Immunolabeling and co-immunoprecipitation studies suggest that iron is stored in ferritin 2 for the purpose of forming 7.5-nm diameter iron particles and that actin-myosin-ferritin 2 may serve as a transporter system. This system, along with calcium and ATP, conveys the iron particles (ferritin) to the center of iron deposition vesicles for iron granules formation. These proteins and reactants are included in iron deposition vesicles during the formation of iron deposition vesicles from the fusion of smooth endoplasmic reticulum. A hypothetical model for magnetite biomineralization in iron deposition vesicles is proposed for honeybees