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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Protein‐protein docking by shape‐complementarity and property matching

We present a computational approach to protein‐protein docking based on surface shape complementarity (“ProBinder”). Within this docking approach, we implemented a new surface decomposition method that considers local shape features on the protein surface. This new surface shape decomposition results in a deterministic representation of curvature features on the protein surface, such as “knobs,” “holes,” and “flats” together with their point normals. For the actual docking procedure, we used geometric hashing, which allows for the rapid, translation‐, and rotation‐free comparison of point coordinates. Candidate solutions were scored based on knowledge‐based potentials and steric criteria. The potentials included electrostatic complementarity, desolvation energy, amino acid contact preferences, and a van‐der‐Waals potential. We applied ProBinder to a diverse test set of 68 bound and 30 unbound test cases compiled from the Dockground database. Sixty‐four percent of the protein‐protein test complexes were ranked with an root mean square deviation (RMSD) < 5 Å to the target solution among the top 10 predictions for the bound data set. In 82% of the unbound samples, docking poses were ranked within the top ten solutions with an RMSD < 10 Å to the target solution. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Synthesis of N,N‐Dialkylamino‐nor‐Dihydroxanthene‐Hemicyanine Fused Near‐Infrared Fluorophores and Their First Water‐Soluble and/or Bioconjugatable Analogues

The effective synthesis of extended conjugated N ,N ‐dialkylamino‐nor ‐dihydroxanthene‐based fluorophores is described from diversely functionalized salicylic aldehydes. The access to these original fluorescent derivatives proceeded in two steps through a one‐pot construction of the unusual nor ‐dihydroxanthene (nor ‐DHX) scaffold followed by a diversification step providing a wide variety of nor ‐DHX‐hemicyanine fused dyes emitting in the range of 730–790 nm. The versatility of our approach has enabled a further extension to the late‐stage introduction of negatively/positively charged polar groups onto their terminal nitrogen heterocyclic subunit, thereby giving access to the first water‐soluble and/or bioconjugatable members of this emerging class of NIR fluorophores. Our water‐solubilizing method is easily implementable, and the nor ‐DHX‐hemicyanine skeleton maintains satisfying fluorescence quantum yields (5–20 %) under physiological conditions. Finally, the bioconjugation ability of fluorescent derivatives bearing a free carboxylic acid was demonstrated through the covalent labeling of a model protein, namely, bovine serum albumin.     

Oxime Side‐Chain Cross‐Links in an α‐Helical Coiled‐Coil Protein: Structure,Thermodynamics, and Folding‐Templated Synthesis of Bicyclic Species

Covalent side‐chain cross‐links are a versatile method to control peptide folding, particularly when α‐helical secondary structure is the target. Here, we examine the application of oxime bridges, formed by the chemoselective reaction between aminooxy and aldehyde side chains, for the stabilization of a helical peptide involved in a protein–protein complex. A series of sequence variants of the dimeric coiled coil GCN4‐p1 bearing oxime bridges at solvent‐exposed positions were prepared and biophysically characterized. Triggered unmasking of a side‐chain aldehyde in situ and subsequent cyclization proceed rapidly and cleanly at pH 7 in the folded protein complex. Comparison of folding thermodynamics among a series of different oxime bridges show that the cross links are consistently stabilizing to the coiled coil, with the extent of stabilization sensitive to the exact size and structure of the macrocycle. X‐ray crystallographic analysis of a coiled coil with the best cross link in place and a second structure of its linear precursor show how the bridge is accommodated into an α‐helix. Preparation of a bicyclic oligomer by simultaneous formation of two linkages in situ demonstrates the potential use of triggered oxime formation to both trap and stabilize a particular peptide folded conformation in the bound state.     

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.     

The reactivity of human serum albumin toward trans‐4‐hydroxy‐2‐nonenal

Mass spectrometry was used to probe the preferred locations of trans‐4‐hydroxy‐2‐nonenal (HNE) addition to the cysteine, histidine, and lysine residues of human serum albumin (HSA). Considering only those modified peptides supported by high mass accuracy Orbitrap precursor ion measurements (high confidence hits), with HNE:HSA ratios of 1:1 and 10:1, 3 and 15 addition sites, respectively, were identified. Using less stringent criteria, a total of 34 modifications were identified at the higher concentration. To gain quantitative data, iTRAQ labeling studies were completed. Previous work had identified Cys³⁴, the only free cysteine, as the most reactive residue in HSA, and we have found that Lys¹⁹⁹, His^242/7, and His²⁸⁸ are the next most reactive residues. Although the kinetic data indicate that the lysines and histidines can react at relatively similar rates, the results show that lysine addition is much less favorable thermodynamically; under our reaction conditions, lysine addition generally does not go to completion. This suggests that under physiological conditions, HNE addition to lysine is only relevant in situations where unusually high HNE concentrations or access to irreversible secondary reactions are found. Copyright © 2012 John Wiley & Sons, Ltd.     

“Head‐to‐Middle” and “Head‐to‐Tail” cis‐Prenyl Transferases: Structure of Isosesquilavandulyl Diphosphate Synthase

We report the first X‐ray crystallographic structure of the “head‐to‐middle” prenyltransferase, isosesquilavandulyl diphosphate synthase, involved in biosynthesis of the merochlorin class of antibiotics. The protein adopts the ζ or cis‐prenyl transferase fold but remarkably, unlike tuberculosinol adenosine synthase and other cis‐prenyl transferases (e.g. cis‐farnesyl, decaprenyl, undecaprenyl diphosphate synthases), the large, hydrophobic side chain does not occupy a central hydrophobic tunnel. Instead, it occupies a surface pocket oriented at 90° to the hydrophobic tunnel. Product chain‐length control is achieved by squeezing out the ligand from the conventional allylic S1 binding site, with proton abstraction being achieved using a diphosphate‐Asn‐Ser relay. The structures revise and unify our thinking as to the mechanism of action of many other prenyl transferases and may also be of use in engineering new merochlorin‐class antibiotics.     

Folding Dynamics of 10‐Residue β‐Hairpin Peptide Chignolin

Short peptides that fold into β‐hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7 μs) of the 10‐residue β‐hairpin peptide chignolin, which is the smallest β‐hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen‐bond network and the complete hydrophobic core as well as the arrangement of side‐chain–side‐chain interactions occur at approximately the same time. This three‐step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the β‐hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa.