Unmodified Polypeptide Research Articles

Size-Dependent Hydrogen Atom Attachment to Gas-Phase Hydrogen-Deficient Polypeptide Radical Cations.

Despite significant affinity to carbonyl oxygens, thermal hydrogen atoms attach to unmodified polypeptides at a very low rate, while the hydrogen-hydrogen exchange rate is high. Here, using the novel omnitrap setup, we found that attachment to polypeptides is much more facile when radical site is already present, but the rate decreases for larger radical ions. The likely explanation is the intramolecular hydrogen atom rearrangement in hydrogen-deficient radicals to a more stable or less accessible site.

Engineering the Aromaticity of Cationic Helical Polypeptides toward "Self-Activated" DNA/siRNA Delivery.

The development of potent yet nontoxic membrane-penetrating materials is in high demand for effective intracellular gene delivery. We have recently developed α-helical polypeptides which afford potent membrane activities to facilitate intracellular DNA delivery via both endocytosis and the nonendocytic "pore formation" mechanism. Endocytosis will cause endosomal entrapment of the DNA cargo, while excessive "pore formation" would cause appreciable cytotoxicity. Additionally, helical polypeptides with stiff, rodlike structure suffer from low siRNA binding affinity. To address such critical issues, we herein incorporated various aromatic domains (benzyl, naphthyl, biphenyl, anthryl, and pyrenyl) into the side-chain terminals of guanidine-rich, helical polypeptides, wherein the flat-rigid shape, π-electronic structures of aromatic motifs "self-activated" the membrane-penetrating capabilities of polypeptides to promote intracellular gene delivery. Benzyl (Bn)- and naphthyl (Naph)-modified polypeptides demonstrated the highest DNA uptake level that outperformed the unmodified polypeptide, P2, by ∼4 fold. More importantly, compared with P2, Bn- and Naph-modified polypeptides allowed more DNA cargos to be internalized via the nonendocytic pathway, which significantly bypassed the endosomal entrapment and accordingly enhanced the transfection efficiency by up to 42 fold, outperforming PEI 25k as the commercial reagent by 3-4 orders of magnitude. The aromatic modification also improved the siRNA condensation capability of polypeptides, achieving notably enhanced gene-silencing efficiency against tumor necrosis factor-α to treat acute hepatic inflammation. Furthermore, we revealed that aromaticity-augmented membrane activity was accompanied by comparable or even significantly reduced "pore formation" capability, thus leading to diminished cytotoxicity at high concentrations. This study therefore provides a promising approach to manipulate the membrane activities and penetration mechanisms of polycations, which overcomes the multiple critical barriers preventing effective and safe gene delivery.

133 Structural basis for ebolavirus matrix assembly and budding; protein plasticity allows multiple functions

Proteins, particularly viral proteins, can be multifunctional, but the mechanism(s) behind this trait are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry and cellular microscopy that the Ebola virus VP40 protein rearranges into different structures, each with a distinct and essential function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer trafficks to the cellular membrane. There, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multi-layered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly, but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle, and demonstrate how a single, wild-type, unmodified polypeptide can assemble into different structures for different functions.

Structural Rearrangement of the Ebola Virus VP40 Protein Begets Multiple Functions in the Virus Life Cycle

Structural basis for ebolavirus matrix assembly and budding; protein plasticity allows multiple functions Proteins, particularly viral proteins, can be multifunctional, but the mechanism(s) behind this trait are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry and cellular microscopy that a protein of the Ebola virus, termed VP40, rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer trafficks to the cellular membrane. There, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multi-layered, filamentous structure that is critical for assembly and budding of nascent virions and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement pathway, is not involved in virus assembly, but instead exists only in infected cells, where it uniquely binds RNA to regulate viral transcription. These results provide a functional model for Ebola virus matrix assembly and the other roles of VP40 in the virus life cycle, and demonstrate how a single, wild-type, unmodified polypeptide can assemble into different structures for different functions.

Structural Rearrangement of Ebola Virus VP40 Begets Multiple Functions in the Virus Life Cycle

Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.

Studies on the Essential Intramolecular Interaction Between the A1 and A2 Domains of von Willebrand Factor

Haemostasis depends on the balanced participation of von Willebrand factor (vWF), a large multimeric and multidomain glycoprotein with essential role during the initial steps of blood clotting. Mature vWF circulates in plasma with the form of multimers comprised of several domains with diverse functions. More specifically, the A1 domain of vWF plays crucial role in haemostasis, regulating the mechanism of platelet adhesion in sites of vascular injury while A2 domain regulates the normal turnover of vWF. Recent studies have implied that an intramolecular interaction between A1 and A2 domains exists, which prevents platelets adhesion and subsequently inhibits the initial step of the blood coagulation mechanism. In an effort to elucidate the essential nature of the interaction between these two domains, we produced and purified the corresponding recombinant unmodified polypeptides. The secondary structure of the two domains was studied individually and as a mixture using circular dichroism spectroscopy. The observed interaction was verified by ELISA competition assays using antibodies and their ability to form productive interactions was further characterized kinetically. In silico analysis (docking and molecular dynamics simulations) of the A1-A2 binding indicated three possible structural models highlighting the crucial, for this interaction, region.

Studies on the Essential Intramolecular Interaction Between the A1 and A2 Domains of von Willebrand Factor

Haemostasis depends on the balanced participation of von Willebrand factor (vWF), a large multimeric and multidomain glycoprotein with essential role during the initial steps of blood clotting. Mature vWF circulates in plasma with the form of multimers comprised of several domains with diverse functions. More specifically, the A1 domain of vWF plays crucial role in haemostasis, regulating the mechanism of platelet adhesion in sites of vascular injury while A2 domain regulates the normal turnover of vWF. Recent studies have implied that an intramolecular interaction between A1 and A2 domains exists, which prevents platelets adhesion and subsequently inhibits the initial step of the blood coagulation mechanism. In an effort to elucidate the essential nature of the interaction between these two domains, we produced and purified the corresponding recombinant unmodified polypeptides. The secondary structure of the two domains was studied individually and as a mixture using circular dichroism spectroscopy. The observed interaction was verified by ELISA competition assays using antibodies and their ability to form productive interactions was further characterized kinetically. In silico analysis (docking and molecular dynamics simulations) of the A1-A2 binding indicated three possible structural models highlighting the crucial, for this interaction, region. Keywords: von Willebrand factor, A1-A2 domain interaction, Circular Dichroism spectroscopy, Molecular Dynamics simulations

Increasing Solubility of Proteins and Peptides by Site-Specific Modification with Betaine

Proteins and peptides with low solubility and which aggregate are often encountered in biochemical studies and in pharmaceutical applications of polypeptides. Here, we report a new strategy to improve solubility and prevent aggregation of polypeptides using site-specific modification with the small molecule betaine, which contains a quaternary ammonium moiety. Betaine was site-selectively attached to the N-termini of two aggregation-prone polypeptide models, the bacterial enzyme xanthine-guanine phosphoribosyltransferase (CG-GPRT) and the HIV entry inhibitor peptide CG-T20, utilizing native chemical ligation. N-terminal cysteines for the betaine ligation reactions were generated from His-tagged fusion proteins using TEV protease cleavage. Ligation of the betaine thioester (1) to the N-terminal cysteine-containing polypeptide models proceeded in high yield, though denaturing conditions were required for CG-T20 due to the hydrophobic nature of this peptide. CD spectroscopy and GPRT activity assays indicate that the betaine modification of CG-GPRT and CG-T20 does not significantly affect structure or activity of the polypeptides. Solubility and turbidity measurements of betaine-modified and unmodified polypeptides demonstrate that betaine modification can greatly increase solubility. Finally, it is shown that betaine-modified CG-T20 acts as an inhibitor of the aggregation of unmodified CG-T20.

Endoplasmic Reticulum Stress Signaling in Disease

The extracellular space is an environment hostile to unmodified polypeptides. For this reason, many eukaryotic proteins destined for exposure to this environment through secretion or display at the cell surface require maturation steps within a specialized organelle, the endoplasmic reticulum (ER). A complex homeostatic mechanism, known as the unfolded protein response (UPR), has evolved to link the load of newly synthesized proteins with the capacity of the ER to mature them. It has become apparent that dysfunction of the UPR plays an important role in some human diseases, especially those involving tissues dedicated to extracellular protein synthesis. Diabetes mellitus is an example of such a disease, since the demands for constantly varying levels of insulin synthesis make pancreatic beta-cells dependent on efficient UPR signaling. Furthermore, recent discoveries in this field indicate that the importance of the UPR in diabetes is not restricted to the beta-cell but is also involved in peripheral insulin resistance. This review addresses aspects of the UPR currently understood to be involved in human disease, including their role in diabetes mellitus, atherosclerosis, and neoplasia.

Post-translational Amino Acid Isomerization: A FUNCTIONALLY IMPORTANT d-AMINO ACID IN AN EXCITATORY PEPTIDE

The post-translational modification of an L- to a D-amino acid has been documented in relatively few gene products, mostly in small peptides under 10 amino acids in length. In this report, we demonstrate that a 46-amino acid polypeptide toxin has one D-phenylalanine at position 44, and that the epimerization from an L-Phe to a D-Phe has a dramatic effect on the excitatory effects of the peptide. In one electrophysiological assay carried out, the D-Phe-containing peptide was extremely potent, whereas the unmodified polypeptide had no biological activity, demonstrating that the chirality of the post-translationally modified amino acid is functionally significant. The peptide toxin analyzed, r11a, belongs to the I-gene superfamily of conotoxins that has four disulfide cross-links. The D-Phe in r11a is at the third amino acid from the C terminus, the same relative position from the C-terminal end as the d-amino acid in omega-agatoxin TK from a spider, an unrelated peptide. Thus, although post-translational amino acid isomerization appears to have no strong specificity for the chemical nature of the amino acid side chain, the few peptides where this modification has been established suggest that there may be favored positions near the N or C terminus that are preferential sites for isomerization to a D-amino acid.

MARKET REPORT PREVIEW: Nonwovens in Filtration Worldwide

Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.

Low Affinity Binding of Interleukin-1β and Intracellular Signaling via NF-κB Identify Fit-1 as a Distant Member of the Interleukin-1 Receptor Family

The fit-1 gene gives rise to two different mRNA isoforms, which code for soluble (Fit-1S) and membrane-bound (Fit-1M) proteins related to the type I interleukin (IL)-1 receptor. To investigate IL-1 binding, we have synthesized and purified histidine-tagged polypeptides corresponding to Fit-1S and the extracellular domain of the type I IL-1 receptor using a vaccinia expression system. Fit-1S is shown to interact with IL-1 beta, but not with IL-1 alpha. However, Fit-1S binds IL-1 beta only with low affinity in contrast to the IL-1 receptor, suggesting that IL-1 beta is not a physiological ligand of Fit-1S. Moreover, expression of the membrane-bound protein Fit-1M in transiently transfected Jurkat cells did not result in activation of the transcription factor NF-kappa B following IL-1 beta treatment. However, a chimeric protein consisting of the extracellular domain of the type I IL-1 receptor and of the transmembrane and intracellular regions of Fit-1M stimulated NF-kappa B-dependent transcription as efficiently as the full-length type I IL-1 receptor. These data indicate that Fit-1M is a signaling molecule belonging to the IL-1 receptor family.

Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein.

We have determined the complete sequence of the cDNA clone representing the full size human skin collagenase mRNA. Collagenase is synthesized in preproenzyme form, Mr 54,092, with a 19 amino acid long signal peptide. The primary secretion products of the enzyme consist of a minor glycosylated form, Mr 57,000, and a major unmodified polypeptide of predicted Mr 51,929. Proteolytic activation of human skin procollagenase results in removal of 81 amino acid residues from the amino-terminal portion of the proenzyme. Both potential N-glycosylation sites are contained within the proteolytically activated form of the enzyme. The primary structure of the coding region of the presented clone is homologous to an oncogene-induced rat protein whose function is still unknown, although preliminary observations suggest that it is not rat skin collagenase.

Reconstructing normal alpha-crystallin from the modified cataractous protein.

alpha-Crystallin from human cataractous lenses is so extensively modified that it is unsuitable for structural studies. We have disassembled the protein and, from the remaining traces of unmodified polypeptides, reconstructed a more homogeneous and conformationally discrete molecule. We suggest that this approach may be particularly useful to studies of enzymology and molecular gerontology.

POLYMORPHIC POSTTRANSLATIONAL MODIFICATION OF ALKALINE PHOSPHATASE IN ESCHERICHIA COLI.

Natural isolates of Escherichia coli have been examined by polyacrylamide gel electrophoresis for variant isozyme patterns of alkaline phosphatase. The polypeptide chains of this enzyme normally exist in two forms-an unmodified polypeptide product of the phoA gene and a posttranslationally modified version of the same polypeptide that has had its N-terminal arginine removed through the action of the iap gene product. These two forms of the polypeptide aggregate as dimers and thus normally form three electrophoretically distinguishable alkaline phosphatase isozymes. Among 104 strains screened, three had variant isozyme patterns. Two of these were deficient in the posttranslationally modified polypeptide, and cotransduction with cysI indicates that they carry mutant iap genes. The third variant is deficient in the unmodified form of the polypeptide. These results provide an unambiguous case of polymorphic posttranslational modification in E. coli.

Interferoids: In vitro and in vivo conversion of native interferons to lower molecular weight forms

Mouse interferons appear as two distinct molecular forms, one migrating at 38,000 daltons in sodium dodecyl sulfate/polyacrylamide gels and one migrating at 22,000 daltons; these interferons comprise about 80% and 20% of total activities, respectively. When such interferon preparations are briefly exposed to acidic periodate buffer, the larger interferon species is apparently converted to the smaller form since the activity at 38,000 daltons is completely eliminated while the activity at 22,000 daltons increases significantly; upon further oxidative cleavage, antiviral activity becomes detectable migrating at 15,000 daltons. Because no native mouse interferon has been reported as such small molecules, this antiviral activity is designated mouse "interferoid" to distinguish it from the native, naturally occurring interferon forms. Prolonged acidperiodate treatment fails to quantitatively convert the 22,000-dalton interferon to the 15,000-dalton interferoid since both are inactivated. When L cells are induced to make interferon in the presence of glycosylation inhibitors, either D-glucosamine or 2-deoxy-D-glucose, they produce approximately normal levels of antiviral activity. However, when such preparations are analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, little activity (<10%) migrates as either the 38,000-dalton or 22,000-dalton native interferons. The interferons and interferoid are antigenically and hydrophobically indistinguishable. These data suggest that induced mouse cells normally synthesize the interferoid as a precursor polypeptide that is either partially or extensively modified by carbohydrate additions to produce, respectively, the 22,000- and 38,000-dalton mouse interferons. Because interferoid is apparently fully biologically active without these moieties, chemical synthesis of such unmodified polypeptides or active fragments from them appears feasible.

On the resolution of polypeptides by isoelectric focusing in polyacrylamide gels in the presence of urea and nonidet-P40

Three model substances were used to test the resolving power of isoelectric focusing in polyacrylamide gels in the presence of 9 m urea and 2% Nonidet-P40: (1) The α subunit of RNA polymerase from Escherichia coli modified by a single covalently attached adenosine 5′-diphosphate-ribose residue can be clearly resolved from the unmodified subunit. (2) The α subunit of RNA polymerase from a mutant of E. coli in which a single leucine residue is replaced by a histidine residue can be resolved from the wild-type subunit. (3) Muscle actin presumably modified by carboxymethylation with iodoacetate at only one cysteine residue can be separated from the unmodified polypeptide.

Reactivity difference between haemoglobins. Part XIX

Reactivity difference between haemoglobins. Part XIX