Convenient Approach to Polypeptide Copolymers Derived from Native Proteins

A convenient approach for the synthesis of narrowly dispersed polypeptide copolymers of defined compositions is presented. The controlled denaturation of the proteins serum albumin and lysozyme followed by an in situ stabilization with polyethylene­(oxide) chains yields polypeptide side chain copolymers of precisely defined backbone lengths as well as the presence of secondary structure elements. Supramolecular architectures are formed in solution because of the presence of hydrophobic and hydrophilic amino acids along the polypeptide main chain. Polypeptide copolymers reported herein reveal excellent solubility and stability in aqueous media and no significant cytotoxicity at relevant concentrations, and they can be degraded via proteolysis, which is very attractive for biomedical applications. This “semi-synthetic chemistry” approach is based on a novel and convenient concept for producing synthetic polypeptides from native protein resources, which complements traditional polypeptide synthesis and expression approaches and offers great opportunities for the preparation of diverse polypeptides with unique architectures

Self-Assembly of High Molecular Weight Polypeptide Copolymers Studied via Diffusion Limited Aggregation

The assembly of high molecular weight polypeptides into complex architectures exhibiting structural complexity ranging from the nano- to the mesoscale is of fundamental importance for various protein-related diseases but also hold great promise for various nano- and biotechnological applications. Here, the aggregation of partially unfolded high molecular weight polypeptides into multiscale fractal structures is investigated by means of diffusion limited aggregation and atomic force microscopy. The zeta potential, the hydrodynamic radius, and the obtained fractal morphologies were correlated with the conformation of the polypeptide backbones as obtained from circular dichroism measurements. The polypeptides are modified with polyethylene oxide side chains to stabilize the polypeptides and to normalize intermolecular interactions. The modification with the hydrophobic thioctic acid alters the folding of the polypeptide backbone, resulting in a change in solution aggregation and fractal morphology. We found that a more compact folding results in dense and highly branched structures, whereas a less compact folded polypeptide chain yields a more directional assembly. Our results provide first evidence for the role of compactness of polypeptide folding on aggregation. Furthermore, the mesoscale-structured biofilms were used to achieve a hierarchical protein assembly, which is demonstrated by deposition of Rhodamine-labeled HSA with the preassembled fractal structures. These results contribute important insights to the fundamental understanding of the aggregation of high molecular weight polypeptides in general and provide opportunities to study nanostructure-related effects on biological systems such as adhesion, proliferation, and the development of, for example, neuronal cells

pH responsive supramolecular core-shell protein hybrids

<p>PEGylation of proteins remains an integral part of macromolecular therapeutics due to its well-known benign effects and pharmacokinetic enhancement properties. We report herein that PEGylation can be taken to the next level of complexity and dynamic behaviour by introducing highly stable but responsive supramolecular handles. By attaching small boronic acid groups onto proteins and salicylhydroxamate moiety to end-functionalise PEG chains, we demonstrate a comprehensive study on the facile assembly/disassembly of a core-shell protein–polymer architecture using fluorescence and microscale thermophoresis on a macromolecular level. In addition, we demonstrate that both the activity and cellular transfer of functional proteins remained conserved throughout the assembly process thus establishing a rapid and orthogonal strategy towards protein PEGylation.</p

Copper-Catalyzed Regioselective Intramolecular Electrophilic Sulfenoamination via Lewis Acid Activation of Disulfides under Aerobic Conditions

The activation of disulfides by Cu­(II) salts has been realized, which triggers a highly efficient electrophilic sulfenoamination of alkenes under aerobic conditions. Various sulfenyl N-heterocycles and their Selena counterparts were produced regioselectively, with no competing disulfidation products detected. Mechanistic studies suggest a profound influence of the counterions on the Lewis acidic copper center, and the important roles of oxygen and DMSO as co-oxidants for these cyclization processes

DataSheet1.PDF

<p>Transfer cells (TCs) play important roles in facilitating enhanced rates of nutrient transport at key apoplasmic/symplasmic junctions along the nutrient acquisition and transport pathways in plants. TCs achieve this capacity by developing elaborate wall ingrowth networks which serve to increase plasma membrane surface area thus increasing the cell's surface area-to-volume ratio to achieve increased flux of nutrients across the plasma membrane. Phloem parenchyma (PP) cells of Arabidopsis leaf veins trans-differentiate to become PP TCs which likely function in a two-step phloem loading mechanism by facilitating unloading of photoassimilates into the apoplasm for subsequent energy-dependent uptake into the sieve element/companion cell (SE/CC) complex. We are using PP TCs in Arabidopsis as a genetic model to identify transcription factors involved in coordinating deposition of the wall ingrowth network. Confocal imaging of pseudo-Schiff propidium iodide-stained tissue revealed different profiles of temporal development of wall ingrowth deposition across maturing cotyledons and juvenile leaves, and a basipetal gradient of deposition across mature adult leaves. RNA-Seq analysis was undertaken to identify differentially expressed genes common to these three different profiles of wall ingrowth deposition. This analysis identified 68 transcription factors up-regulated two-fold or more in at least two of the three experimental comparisons, with six of these transcription factors belonging to Clade III of the NAC-domain family. Phenotypic analysis of these NAC genes using insertional mutants revealed significant reductions in levels of wall ingrowth deposition, particularly in a double mutant of NAC056 and NAC018, as well as compromised sucrose-dependent root growth, indicating impaired capacity for phloem loading. Collectively, these results support the proposition that Clade III members of the NAC-domain family in Arabidopsis play important roles in regulating wall ingrowth deposition in PP TCs.</p

Spatiotemporally Controlled Photolabeling of Genetically Unmodified Proteins in Live Cells

Selective labeling of the protein of interest (POI) in genetically unmodified live cells is crucial for understanding protein functions and kinetics in their natural habitat. In particular, spatiotemporally controlled installation of the labels on a POI under light control without affecting their original activity is in high demand but is a tremendous challenge. Here, we describe a novel ligand-directed photoclick strategy for spatiotemporally controlled labeling of endogenous proteins in live cells. It was realized with a designer labeling reagent skillfully integrating the photochemistries of 2-nitrophenylpropyloxycarbonyl and 3-hydroxymethyl-2-naphthol with an affinity ligand. Highly electrophilic ortho-naphthoquinone methide was photochemically released and underwent a proximity coupling reaction with nucleophilic amino acid residues on the POI in live cells. With fluorescein as a marker, this photoclick strategy enables time-resolved labeling of carbonic anhydrase subtypes localized either on the cell membrane or in the cytoplasm and a discriminable visualization of their metabolic kinetics. Given the versatility underlined by facilely tethering other functional entities (e.g., biotin, a peptide short chain) via acylation or (in cell) Huisgen cycloaddition, this affinity-driven photoclick chemistry opens up enormous opportunities for discovering dynamic functions and mechanistic interrogation of endogenous proteins in live cells

pH Responsive Janus-like Supramolecular Fusion Proteins for Functional Protein Delivery

A facile, noncovalent solid-phase immobilization platform is described to assemble Janus-like supramolecular fusion proteins that are responsive to external stimuli. A chemically postmodified transporter protein, DHSA, is fused with (imino)­biotinylated cargo proteins via an avidin adaptor with a high degree of spatial control. Notably, the derived heterofusion proteins are able to cross cellular membranes, dissociate at acidic pH due to the iminobiotin linker and preserve the enzymatic activity of the cargo proteins β-galactosidase and the enzymatic subunit of <i>Clostridium botulinum</i> C2 toxin. The mix-and-match strategy described herein opens unique opportunities to access macromolecular architectures of high structural definition and biological activity, thus complementing protein ligation and recombinant protein expression techniques

Dendronized Albumin Core–Shell Transporters with High Drug Loading Capacity

We describe the synthesis of a core–shell biohybrid consisting of a human serum albumin (HSA) core that serves as a reservoir for lipophilic molecules and a cationized shell region consisting of <b>ethynyl-G2.0</b>-<b>PAMAM</b> or <b>ethynyl-G3.0</b>-<b>PAMAM</b> dendrons. The binding capacity of lipophilic guests was quantified applying electron paramagnetic resonance (EPR) spectroscopy, and five to six out of seven pockets were still available compared with HSA. The attachment of <b>ethynyl-G2.0</b>-<b>PAMAM</b> dendrons to HSA yielded a nontoxic core–shell macromolecule that was clearly uptaken by A549 human epithelial cells due to the presence of the dendritic PAMAM shell. Significantly higher loading of doxorubicin was observed for dendronized <b>G2-DHSA</b> compared with the native protein due to the availability of binding pockets of the HSA core, and interaction with the dendritic shell. Dendronized <b>G2-DHSA</b>-doxorubicin displayed significant cytotoxicity resulting from high drug loading and high stability under different conditions, thus demonstrating its great potential as a transporter for drug molecules

DNA-Based Self-Assembly of Fluorescent Nanodiamonds

As a step toward deterministic and scalable assembly of ordered spin arrays we here demonstrate a bottom-up approach to position fluorescent nanodiamonds (NDs) with nanometer precision on DNA origami structures. We have realized a reliable and broadly applicable surface modification strategy that results in DNA-functionalized and perfectly dispersed NDs that were then self-assembled in predefined geometries. With optical studies we show that the fluorescence properties of the nitrogen-vacancy color centers in NDs are preserved during surface modification and DNA assembly. As this method allows the nanoscale arrangement of fluorescent NDs together with other optically active components in complex geometries, applications based on self-assembled spin lattices or plasmon-enhanced spin sensors as well as improved fluorescent labeling for bioimaging could be envisioned