A Synthetic Adenylation‐Domain‐Based tRNA‐Aminoacylation Catalyst

The incorporation of non‐proteinogenic amino acids represents a major challenge for the creation of functionalized proteins. The ribosomal pathway is limited to the 20–22 proteinogenic amino acids while nonribosomal peptide synthetases (NRPSs) are able to select from hundreds of different monomers. Introduced herein is a fusion‐protein‐based design for synthetic tRNA‐aminoacylation catalysts based on combining NRPS adenylation domains and a small eukaryotic tRNA‐binding domain (Arc1p‐C). Using rational design, guided by structural insights and molecular modeling, the adenylation domain PheA was fused with Arc1p‐C using flexible linkers and achieved tRNA‐aminoacylation with both proteinogenic and non‐proteinogenic amino acids. The resulting aminoacyl‐tRNAs were functionally validated and the catalysts showed broad substrate specificity towards the acceptor tRNA. Our strategy shows how functional tRNA‐aminoacylation catalysts can be created for bridging the ribosomal and nonribosomal worlds. This opens up new avenues for the aminoacylation of tRNAs with functional non‐proteinogenic amino acids.     

Induced Folding of Protein‐Sized Foldameric β‐Sandwich Models with Core β‐Amino Acid Residues

The mimicry of protein‐sized β‐sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin‐14 β‐sheet has been used as a template, and α→β residue mutations were carried out in the hydrophobic core (positions 12 and 19). β‐Residues with diverse structural properties were utilized: Homologous β³‐amino acids, (1R,2S)‐2‐aminocyclopentanecarboxylic acid (ACPC), (1R,2S)‐2‐aminocyclohexanecarboxylic acid (ACHC), (1R,2S)‐2‐aminocyclohex‐3‐enecarboxylic acid (ACEC), and (1S,2S,3R,5S)‐2‐amino‐6,6‐dimethylbicyclo[3.1.1]heptane‐3‐carboxylic acid (ABHC). Six α/β‐peptidic chains were constructed in both monomeric and disulfide‐linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced β‐sheet formation in the 64‐residue foldameric systems. Core replacement with (1R,2S)‐ACHC was found to be unique among the β‐amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen‐bonding network and to fit sterically into the hydrophobic interior of the β‐sandwich. The novel β‐sandwich model containing 25 % unnatural building blocks afforded protein‐like thermal denaturation behavior.     

Design and Applications of Protein‐Cage‐Based Nanomaterials

Materials science is beginning to focus on biotemplation, and in support of that trend, it is realized that protein cages—proteins that assemble from multiple monomers into architectures with hollow interiors—can instill a number of unique advantages to nanomaterials. In addition, the structural and functional plasticity of many protein‐cage systems permits their engineering for specific applications. In this review, the most commonly used viral and non‐viral protein cages, which exhibit a wide diversity of size, functionality, and chemical and thermal stabilities, are described. Moreover, how they have been exploited for nanomaterial and nanotechnology applications is summarized.     

Enzyme‐Mediated,Site‐Specific Protein Coupling Strategies for Surface‐Based Binding Assays

Covalent surface immobilization of proteins for binding assays is typically performed non‐specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4′‐phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site‐specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors’ functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor–ligand pairs.     

Diphenylacetylene‐Linked Peptide Strands Induce Bidirectional β‐Sheet Formation

In the search for synthetic mimics of protein secondary structures relevant to the mediation of protein–protein interactions, we have synthesized a series of tetrasubstituted diphenylacetylenes that display β‐sheet structures in two directions. Extensive X‐ray crystallographic and NMR solution phase studies are consistent with these proteomimetics adopting sheet structures, displaying both hydrophobic and hydrophilic amino acid side chains.     

Self‐Assembled Cage‐Like Protein Structures

Proteins and protein‐based assemblies represent the most structurally and functionally diverse molecules found in nature. Protein cages, viruses and bacterial microcompartments are highly organized structures that are composed primarily of protein building blocks and play important roles in molecular ion storage, nucleic acid packaging and catalysis. The outer and inner surface of protein cages can be modified, either chemically or genetically, and the internal cavity can be used to template, store and arrange molecular cargo within a defined space. Owing to their structural, morphological, chemical and thermal diversity, protein cages have been investigated extensively for applications in nanotechnology, nanomedicine and materials science. Here we provide a concise overview of the most common icosahedral viral and nonviral assemblies, their role in nature, and why they are highly attractive scaffolds for the encapsulation of functional materials.     

Determination of side‐chain‐rotamer and side‐chain and backbone virtual‐bond‐stretching potentials of mean force from AM1 energy surfaces of terminally‐blocked amino‐acid residues,for coarse‐grained simulations of protein structure and folding. I. The method

In this and the accompanying article, we report the development of new physics‐based side‐chain‐rotamer and virtual‐bond‐deformation potentials which now replace the respective statistical potentials used so far in our physics‐based united‐reside UNRES force field for large‐scale simulations of protein structure and dynamics. In this article, we describe the methodology for determining the corresponding potentials of mean force (PMF's) from the energy surfaces of terminally‐blocked amino‐acid residues calculated with the AM1 quantum‐mechanical semiempirical method. The approach is based on minimization of the AM1 energy for fixed values of the angles λ for rotation of the peptide groups about the C^α ··· C^α virtual bonds, and for fixed values of the side‐chain dihedral angles χ, which formed a multidimensional grid. A harmonic‐approximation approach was developed to extrapolate from the energy at a given grid point to other points of the conformational space to compute the respective contributions to the PMF. To test the applicability of the harmonic approximation, the rotamer PMF's of alanine and valine obtained with this approach have been compared with those obtained by using a Metropolis Monte Carlo method. The PMF surfaces computed with the harmonic approximation are more rugged and have more pronounced minima than the MC‐calculated surfaces but the harmonic‐approximation‐and MC‐calculated PMF values are linearly correlated. The potentials derived with the harmonic approximation are, therefore, appropriate for UNRES for which the weights (scaling factors) of the energy terms are determined by force‐field optimization for foldability. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Micelle‐Triggered β‐Hairpin to α‐Helix Transition in a 14‐Residue Peptide from a Choline‐Binding Repeat of the Pneumococcal Autolysin LytA

Choline‐binding modules (CBMs) have a ββ‐solenoid structure composed of choline‐binding repeats (CBR), which consist of a β‐hairpin followed by a short linker. To find minimal peptides that are able to maintain the CBR native structure and to evaluate their remaining choline‐binding ability, we have analysed the third β‐hairpin of the CBM from the pneumococcal LytA autolysin. Circular dichroism and NMR data reveal that this peptide forms a highly stable native‐like β‐hairpin both in aqueous solution and in the presence of trifluoroethanol, but, strikingly, the peptide structure is a stable amphipathic α‐helix in both zwitterionic (dodecylphosphocholine) and anionic (sodium dodecylsulfate) detergent micelles, as well as in small unilamellar vesicles. This β‐hairpin to α‐helix conversion is reversible. Given that the β‐hairpin and α‐helix differ greatly in the distribution of hydrophobic and hydrophilic side chains, we propose that the amphipathicity is a requirement for a peptide structure to interact and to be stable in micelles or lipid vesicles. To our knowledge, this “chameleonic” behaviour is the only described case of a micelle‐induced structural transition between two ordered peptide structures.     

A Protein‐Based Pentavalent Inhibitor of the Cholera Toxin B‐Subunit

Protein toxins produced by bacteria are the cause of many life‐threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site‐specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC₅₀) of 104 pM for the CT B‐subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies.     

Construction of a Triangle‐Shaped Trimer and a Tetrahedron Using an α‐Helix‐Inserted Circular Permutant of Cytochrome c555

Highly‐ordered protein structures have gained interest for future uses for biomaterials. Herein, we constructed a building block protein (BBP) by the circular permutation of the hyperthermostable Aquifex aeolicus cytochrome (cyt) c₅₅₅, and assembled BBP into a triangle‐shaped trimer and a tetrahedron. The angle of the intermolecular interactions of BBP was controlled by cleaving the domain‐swapping hinge loop of cyt c₅₅₅ and connecting the original N‐ and C‐terminal α‐helices with an α‐helical linker. We obtained BBP oligomers up to ≈40 mers, with a relatively large amount of trimers. According to the X‐ray crystallographic analysis of the BBP trimer, the N‐terminal region of one BBP molecule interacted intermolecularly with the C‐terminal region of another BBP molecule, resulting in a triangle‐shaped structure with an edge length of 68 Å. Additionally, four trimers assembled into a unique tetrahedron in the crystal. These results demonstrate that the circular permutation connecting the original N‐ and C‐terminal α‐helices with an α‐helical linker may be useful for constructing organized protein structures.     

A Green BODIPY‐Based,Super‐Fluorogenic,Protein‐Specific Labelling Agent

We report the development of YC23, a novel green BODIPY‐based dimaleimide derivative that undergoes a fluorogenic addition reaction (FlARe) with a genetically encodable peptide tag (dC10α) that can be fused to a protein of interest (POI). We also demonstrate the application of this reaction for the fluorogenic labelling of a specific POI in bacterial lysate and in living mammalian cells.     

A novel preparation method for microspheres by glycerol modified solid‐in‐oil‐in‐water multi‐emulsion

Biodegradable material poly(D, L ‐lactic‐co‐glycolic) acid (PLGA) plays an important role in drug‐sustained release systems. Here, we describe a glycerol modified solid‐in‐oil‐in‐water (m‐S/O/W) emulsion method for PLGA microspheres, in order to encapsulate proteins in PLGA by utilizing dextran glassy particles to protect the proteins from denaturing, unfolding, and aggregation during preparation and new external water phase to prevent the inner dextran glassy particles from leaking into the external water phase. External water phase containing 20, 40, 60, 80% glycerol showed that proteins released faster and more completely with increased glycerol content. According to their varied release profiles, microspheres of different formulations could be used to encapsulate vaccines or for delivering proteins over long‐term. Copyright © 2009 John Wiley & Sons, Ltd.