Martin Humenik

C‐Terminal Incorporation of Bio‐Orthogonal Azide Groups into a Protein and Preparation of Protein–Oligodeoxynucleotide Conjugates by CuI‐Catalyzed Cycloaddition

Protein–oligodeoxynucleotide conjugates can be utilized for a variety of applications, like the preparation of synthetic enzymes, gene therapy, electrical DNA microarrays, molecular-scale devices, and the development of immunological assays. A number of protein conjugation techniques have been developed; however, these are usually based on the reaction with functional groups common in biological systems. They therefore lead to random chemical modification and functional heterogeneity of the products. 8] Known bio-orthogonal conjugation reactions, such as Staudinger ligations, ketone/aldehyde-hydrazine reactions, and Cu-catalyzed [3+2] cycloadditions, allow regioand chemoselective modification of proteins, and are especially attractive for construction of protein microarrays, for which function can be significantly altered by protein orientation on the solid surface. 14] Recently we reported the application of an E. coli in vitro transcription–translation coupled system (IVT system) to the site-specific modification of proteins. This was achieved by deactivation of release factor 1 (RF1) by using polyclonal antibodies that led to puromycin incorporation into the C terminus of esterase 2 (EST2). The attachment of nucleotides to the 5’-OH of puromycin was also possible. However, when more then six nucleotides were added to the 5’-OH of puromycin, the reaction yield strongly decreased. This hindered the preparation of protein conjugates with oligodeoxynucleotide (ODN) sufficiently long for specific hybridization. Here, we describe an alternative and equally efficient method for C-terminal modification of proteins by using an E. coli IVT system for incorporation of a bio-orthogonal azide group. With this system 5’-alkyne modified ODNs can be conjugated specifically to the C terminus of a polypeptide chain by Cu-catalyzed cycloaddition via a puromycin linker. Electrochemical detection of protein–ODN conjugates was realized by hybridization to complementary ODNs immobilized on a gold electrode microarray. 2’-Deoxy-cytidylyl-(3’!5’)-puromycin (1; Figure 1A) modified by an NH2 linker at the N4 position of cytidine was used as a starting material for further modifications. For this, N-hydroxy-

Rapid, specific and sensitive electrochemical detection of foodborne bacteria.

Electrochemical biochips are an emerging tool for point-of-care diagnostic systems in medicine, food and environmental monitoring. In the current study, a thermostable reporter enzyme, esterase 2 (EST2) from Alicyclobacillus acidocaldarius, is used for specific and sensitive detection of bacteria by one-step rRNA/DNA hybridization between a bacterium-specific capture oligodeoxynucleotide (ODN), bacterial 16S rRNA and an uniform EST2-ODN reporter conjugate. The detection limit corresponds to approximately 500 colony forming units (cfu) Escherichia coli. Beside high sensitivity, the application of electrochemical biochips allows discrimination of two gram-negative and two gram-positive bacteria demonstrating the specificity and the potential for parallel detection of microorganisms. The feasibility of identification of foodborne bacteria was studied with meat juice contaminated with E. coli. This detection system has the capability to be applied for monitoring of bacterial food contamination.

Self-assembly of nucleic acids, silk and hybrid materials thereof

Top-down approaches based on etching techniques have almost reached their limits in terms of dimension. Therefore, novel assembly strategies and types of nanomaterials are required to allow technological advances. Self-assembly processes independent of external energy sources and unlimited in dimensional scaling have become a very promising approach. Here,we highlight recent developments in self-assembled DNA-polymer, silk-polymer and silk-DNA hybrids as promising materials with biotic and abiotic moieties for constructing complex hierarchical materials in ‘bottom-up’ approaches. DNA block copolymers assemble into nanostructures typically exposing a DNA corona which allows functionalization, labeling and higher levels of organization due to its specific addressable recognition properties. In contrast, self-assembly of natural silk proteins as well as their recombinant variants yields mechanically stable β-sheet rich nanostructures. The combination of silk with abiotic polymers gains hybrid materials with new functionalities. Together, the precision of DNA hybridization and robustness of silk fibrillar structures combine in novel conjugates enable processing of higher-order structures with nanoscale architecture and programmable functions.

Spider Silk: Understanding the Structure–Function Relationship of a Natural Fiber

Spider silk is of great interest because of its extraordinary physical properties, such as strength and toughness. Here we discuss how these physical properties relate to the way in which spiders have utilized this material in prey capture, forcing its evolution to a high-performance fiber. Female spiders can produce up to seven different types of silk, and all these have different physical properties, which relate to their various functions. The variation in properties are due to underlying differences in the proteins making up these silks. As our understanding of spider silk has increased in the recent years, it has been possible to produce recombinant versions of the respective proteins. Recombinant proteins open up the potential to produce synthetic silk fibers with properties similar to those of the natural spider silk threads.

Influence of repeat numbers on self-assembly rates of repetitive recombinant spider silk proteins

Assembly of recombinant spider silk variants eADF4(Cn) comprising different numbers (n) of the consensus sequence motif C, derived from the natural Araneus diadematus dragline silk ADF4, yielded indistinguishable nanofibrils in cases of n⩾2. The C-module comprises 35 amino acids rich in glycine and proline residues (in GPGXY repeats) and one polyalanine stretch (Ala)8. All variants were found to be intrinsically disordered in solution, and upon fibril formation they converted into a cross-β structure. Heterologous seeding indicated high structural compatibility between the different eADF4(Cn) variants, however, their assembly kinetics differed in dependence of the number of repeats. Kinetic analysis revealed a nucleation-growth mechanism typical for the formation of cross-β-fibrils, with nucleation rates as well as growth rates increasing with increasing numbers of repeats. Strikingly, the single C-module did not self-assemble into fibrils, but upon addition of heterologous seeds fibril growth could be observed. Apparently, interconnecting of at least two C-modules significantly facilitates the structural transformation from a disordered state into β-sheet structures, which is necessary for nucleation and beneficial for fibril growth.

Detection of bacterial 16S rRNA using multivalent dendrimer-reporter enzyme conjugates

Novel enzyme-oligodeoxynucleotide conjugate was synthesized to improve sensitivity of Escherichia coli 16S rRNA detection on gold electrodes. Thermostable esterase 2 from Alicyclobacillus acidocaldarius was multiply conjugated to a polyamidoamine dendrimer functionalized by one universal detector oligodeoxynucleotide. Three components rRNA/DNA hybridization between capture oligodeoxynucleotide covalently immobilized on a gold electrode, 16S rRNA and the multivalent esterase-dendrimer cluster was used for detection of E. coli. The linear dependence of the electrochemical signals to analyte concentration revealed a detection limit of 50 colony forming units E. coli, which represents a tenfold signal enhancement if compared to the detection limit achieved with monovalent esterase-oligodeoxynucleotide conjugate.

Ligand‐Directed Immobilization of Proteins through an Esterase 2 Fusion Tag Studied by Atomic Force Microscopy

Atomically flat mica surfaces were chemically modified with an alkyl trifluoromethyl ketone, a covalent inhibitor of esterase 2 from Alicyclobacillus acidocaldarius, which served as a tag for ligand‐directed immobilization of esterase‐linked proteins. Purified NADH oxidase from Thermus thermophilus and human exportin‐t from cell lysates were anchored on the modified surfaces. The immobilization effectiveness of the proteins was studied by atomic force microscopy (AFM). It was shown that ligand–esterase interaction allowed specific attachment of exportin‐t and resulted in high‐resolution images and coverage patterns that were comparable with immobilized purified protein. Moreover, the biological functionality of immobilized human exportin‐t in forming a quaternary complex with tRNA and the GTPase Ran‐GTP, and the dimension changes before and after complex formation were also determined by AFM.

Ion and seed dependent fibril assembly of a spidroin core domain

Recombinant eADF4(C16) represents an engineered spider silk variant based on the sequence of the core domain of the natural dragline silk protein ADF4 of Araneus diadematus. Previously eADF4(C16) has been shown to self-assemble into cross-β fibrils in a two-step process of nucleus formation and fibril growth. Here, it is shown that structurally converted low molecular weight oligomers can act as nuclei. Further, it could be determined that specifically potassium and phosphate ions strongly influence both nucleus formation as well as fibril growth. Nucleation of fibril assembly could be surpassed by seeding soluble protein with pre-assembled fibrils but also, unexpectedly, with eADF4(C16) sub-micrometer particles. The latter finding reveals that spider silk fibril assembly seems to be rather dependent on the protein sequence than on the structural features, since cross-seeding with other proteins was not possible.

Data for ion and seed dependent fibril assembly of a spidroin core domain

This data article includes size exclusion chromatography data of soluble eADF4(C16), an engineered spider silk variant based on the core domain sequence of the natural dragline silk protein ADF4 of Araneus diadematus, in combination with light scattering; the protein is monomeric before assembly. The assembled mature fibrils were visualized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Sonicated fibrils were used as seeds to by-pass the nucleation lag phase in eADF4(C16) assembly. We also provide data on the sedimentation kinetics of spider silk in the presence of different NaCl concentrations revealing very slow protein aggregation in comparison to the fast assembly triggered by phosphate ions published previously [1]. Experiments in the Data article represent supporting material for our work published recently [1], which described the assembly mechanism of recombinant eADF4(C16) fibrils.

Silk nanofibril self-assembly versus electrospinning

Natural silk fibers represent one of the most advanced blueprints for (bio)polymer scientists, displaying highly optimized mechanical properties due to their hierarchical structures. Biotechnological production of silk proteins and implementation of advanced processing methods enabled harnessing the potential of these biopolymer not just based on the mechanical properties. In addition to fibers, diverse morphologies can be produced, such as nonwoven meshes, films, hydrogels, foams, capsules and particles. Among them, nanoscale fibrils and fibers are particularly interesting concerning medical and technical applications due to their biocompatibility, environmental and mechanical robustness as well as high surface-to-volume ratio. Therefore, we introduce here self-assembly of silk proteins into hierarchically organized structures such as supramolecular nanofibrils and fabricated materials based thereon. As an alternative to self-assembly, we also present electrospinning a technique to produce nanofibers and nanofibrous mats. Accordingly, we introduce a broad range of silk-based dopes, used in self-assembly and electrospinning: natural silk proteins originating from natural spinning glands, natural silk protein solutions reconstituted from fibers, engineered recombinant silk proteins designed from natural blueprints, genetic fusions of recombinant silk proteins with other structural or functional peptides and moieties, as well as hybrids of recombinant silk proteins chemically conjugated with nonproteinaceous biotic or abiotic molecules. We highlight the advantages but also point out drawbacks of each particular production route. The scope includes studies of the natural self-assembly mechanism during natural silk spinning, production of silk fibrils as new nanostructured non-native scaffolds allowing dynamic morphological switches, as well as studying potential applications. This article is categorized under:  Biology-Inspired Nanomaterials > Peptide-Based Structures  Nanotechnology Approaches to Biology > Nanoscale Systems in Biology  Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.