Search

The chemical modification of proteins is an effective technique for manipulating the properties and functions of proteins, and for creating protein‐based materials. The N‐terminus is a promising target for single‐site modification that provides modified proteins with uniform structures and properties. In this paper, a copper(II)‐mediated aldol reaction with 2‐pyridinecarboxaldehyde (2‐PC) derivatives is proposed as an operationally simple method to selectively modify the N‐terminus of peptides and proteins at room temperature and physiological pH. The copper(II) ion activates the N‐terminal amino acids by complexation with an imine of the N‐terminal amino acid and 2‐PCs, realizing the selective formation of the nucleophilic intermediate at the N‐terminus. This results in a stable carbon‐carbon bond between the 2‐PCs and the α‐carbon of various N‐terminal amino acids. The reaction is applied to four different proteins, including biopharmaceuticals such as filgrastim and trastuzumab. The modified trastuzumab retains the human epidermal growth factor receptor 2 recognition activity. [ABSTRACT FROM AUTHOR]

Trityl radicals (TAMs) have recently appeared as an alternative source of spin labels for measuring long distances in biological systems. Finland trityl radical (FTAM) served as the basis for this new generation of spin labels, but FTAM is rather lipophilic and susceptible to self‐aggregation, noncovalent binding with lipophilic sites of proteins, and noncovalent docking at the termini of duplex DNA. In this paper the very hydrophilic OX063 TAM with very low toxicity and little tendency for aggregation is used as the basis for a spin label. Human serum albumin (HSA) labeled with OX063 has an intense narrow line typical of TAM radicals in solution, whereas HSA labeled with FTAM shows broad lines and extensive aggregation. In pulse EPR measurements, the measured phase memory time TM for HSA labeled with OX063 is 6.3 μs at 50 K, the longest yet obtained with a TAM‐based spin label. The lowered lipophilicity also decreases side products in the labeling reaction. [ABSTRACT FROM AUTHOR]

The application of computational methods to predict mutations that can enhance the catalytic power of enzymes can be of great importance, since it allows a more rational and directed way to mutate the enzymes, which can save time and resources compared to producing mutations at random. We present computational methods to predict mutations that enhance the catalytic rate of DszD, an enzyme that intervenes in the 4S pathway together with DszA, DszB and DszC. This has potential as a cheaper and greener alternative for biodesulfurization of crude oil. More information can be found in the Full Paper by Ramos et al. on page 17231 ff. [ABSTRACT FROM AUTHOR]

Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X‐ray crystallography. Coating a protein with PEG may have structural consequences, but no single biophysical methodology can spot the possible modifications. This limitation has been overcome by the integration of solution and solid‐state NMR data, relaxometry, and X‐ray crystallography for the characterization of a PEGylated enzyme used in clinical practice. [ABSTRACT FROM AUTHOR]

Nanofabrication based on biological templates has attracted considerable interest because of its applications in materials science and biomedicine. Herein, a facile method is reported for the synthesis of well‐defined TiO2 nanofibers by using a simple N‐(9‐fluorenylmethoxycarbonyl)‐protected phenylalanine–phenylalanine–aspartic acid tripeptide (Fmoc‐Phe–Phe–Asp‐OH, Fmoc‐FFD) as the template. Compared with other synthetic methods of inorganic nanomaterials, these self‐assembling peptides could control the structure and the catalytic activity of the synthesized nanomaterials produced under mild synthetic conditions. The as‐synthesized peptide‐TiO2 hybrid nanofibers and the calcined TiO2 nanofibers were characterized by using transmission electron microscopy (TEM), scanning electron microscopy (SEM), circular dichroism (CD) spectroscopy, and X‐ray diffraction (XRD). The results show that well‐defined TiO2 nanofibers could form when the pH of the peptide solution was 5–7, whereas nanoparticles formed when the pH was 8. Moreover, the peptide‐templated TiO2 nanofibers showed improved photocatalytic activity for the degradation of methyl orange (MO). Finally, the photocatalytic activity of the TiO2 could be controlled by adjusting the pH of the peptide solution during the synthetic process. Peptide template: TiO2 nanomaterials with tunable photocatalytic activity were prepared by using N‐(9‐fluorenylmethoxycarbonyl)‐protected phenylalanine—aspartic acid tripeptide (Fmoc‐FFD) as the template. By incorporating titanium diisopropoxide bis(acetyl‐acetonate) into the peptide solutions, the TiO2 precursors could mineralize and condense with the peptide assemblages. After removing the peptide templates by calcination, a series of TiO2 nanostructures with enhanced photocatalytic activity were formed. [ABSTRACT FROM AUTHOR]

Bioorthogonal reactions have emerged as a versatile tool in life sciences. The inverse electron demand Diels–Alder reaction (DAinv) stands out due to the availability of reactants with very fast kinetics. However, highly reactive dienophiles suffer the disadvantage of being less stable and prone to side reactions. Herein, we evaluate the extent of acceleration of rather unreactive but highly stable dienophiles by DNA-templated proximity. To this end, we developed a modular synthetic route for a novel bifunctional fluorogenic tetrazine rhodamine probe that we used to determine the reaction kinetics of various dienophiles in a fluorescence assay. Under proximity-driven conditions the reaction was found to be several orders of magnitude faster, and we observed almost no background reaction when proximity was not induced. This fundamental study identifies a minimally sized fluorogenic tetrazine dienophile reactant pair that has potential to be generally used for the visualization of biomolecular interactions with temporal and spatial resolution in living systems. [ABSTRACT FROM AUTHOR]

The enhancement of the catalytic power of enzymes is a subject of enormous interest both for science and for industry. The latter, in particular, due to the vast applications enzymes can have in industrial processes, for instance in the desulfurization of crude oil, which is mandatory by law in many developed countries and is currently performed using costly chemical processes. In this work we sought to enhance the turnover rate of DszD from Rhodococcus erythropolis, a NADH-FMN oxidoreductase responsible for supplying FMNH2 to DszA and DszC in the biodesulfurization process of crude oil, the 4S pathway. For this purpose, we replaced the wild type spectator residue of the rate limiting step of the reduction of FMN to FMNH2, a process catalysed by DszD and known to play an important role in the reaction energy profile. As replacements, we used all the naturally occurring amino acids, one at a time, using computational methodologies, and repeated the above-mentioned reaction with each mutant. To calculate the different free energy profiles, one for each mutated model, we applied quantum mechanics/molecular mechanics (QM/MM) methods within an ONIOM scheme. The free energy barriers obtained varied between 15.1 and 29.9 kcal mol−1. Multiple factors contributed to the different Δ G values. The most relevant were electrostatic interactions and the induction of a favourable alignment between substrate and cofactor. These results confirm the great potential that chirurgic mutations have for increasing the catalytic power of DszD in relation to the wild type ( wt) enzyme. [ABSTRACT FROM AUTHOR]

Confinement of reactive chemical species uniquely affects chemical reactivity by restricting the physical space available and by restricting access to interactions with the solvent. In Nature, for example, confined protein binding pockets govern processes following photoisomerization reactions and the isomerizations themselves. Here we describe the first example of a dihydroazulene/vinylheptafulvene (DHA/VHF) photo-switch functioning in water, and we show how its switching behavior is strongly influenced by supramolecular interactions with a series of cucurbit[ n]uril (CB) host molecules. In CB7 inclusion complexes, the kinetics of the thermal VHF-to-DHA back-reaction is accelerated, while in CB8 inclusion complexes, the kinetics is slowed down as compared to the free photo-switch. The effect of the CB encapsulation of the photo-switch can be effectively canceled by introducing a guest that binds the CB more strongly. According to DFT calculations, a stabilization of the reactive s- cis VHF conformer relative to the s- trans VHF appears to be a contributing factor responsible for the accelerated back-reaction when encapsulated in CB7. [ABSTRACT FROM AUTHOR]

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum (ER) resident isoform of the 90 kDa heat shock protein (Hsp90) family and its inhibition represents a promising therapeutic target for the treatment of many diseases. Modification of the first generation cis-amide bioisostere imidazole to alter the angle between the resorcinol ring and the benzyl side chain via cis-amide replacements produced compounds with improved Grp94 affinity and selectivity. Structure-activity relationship studies led to the discovery of compound 30, which exhibits 540 n m affinity and 73-fold selectivity towards Grp94. Grp94 is responsible for the maturation and trafficking of proteins associated with cell signaling and motility, including select integrins. The Grp94-selective inhibitor 30 was shown to exhibit potent anti-migratory effects against multiple aggressive and metastatic cancers. [ABSTRACT FROM AUTHOR]

The productive exploration of chemical space is an enduring challenge in chemical biology and medicinal chemistry. Natural products are biologically relevant, and their frameworks have facilitated chemical tool and drug discovery. A 'top-down' synthetic approach is described that enabled a range of complex bridged intermediates to be converted with high step efficiency into 26 diverse sp3-rich scaffolds. The scaffolds have local natural product-like features, but are only distantly related to specific natural product frameworks. To assess biological relevance, a set of 52 fragments was prepared, and screened by high-throughput crystallography against three targets from two protein families (ATAD2, BRD1 and JMJD2D). In each case, 3D fragment hits were identified that would serve as distinctive starting points for ligand discovery. This demonstrates that frameworks that are distantly related to natural products can facilitate discovery of new biologically relevant regions within chemical space. [ABSTRACT FROM AUTHOR]

The aggregation of amyloidogenic proteins is often related to the occurrence of neurodegenerative diseases, including fused in sarcoma protein (FUS) in frontotemporal lobar degeneration and amyotrophic lateral sclerosis diseases. Recently, the SERF protein family has been reported to have a significant regulatory effect on amyloid formation, but it is still unclear about the detailed mechanisms of SERF acting on different amyloidogenic proteins. Herein, nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy were used to explore interactions of ScSERF with three amyloidogenic proteins FUS‐LC, FUS‐Core, and α‐Synuclein. NMR chemical shift perturbations reveal them sharing similar interaction sites on the N‐terminal region of ScSERF. However, the amyloid formation of α‐Synuclein protein is accelerated by ScSERF, while ScSERF inhibits fibrosis of FUS‐Core and FUS‐LC proteins. Both the primary nucleation and the total amount of fibrils produced are detained. Our results suggest a diverse role of ScSERF in regulating the fibril growth of amyloidogenic proteins. [ABSTRACT FROM AUTHOR]

Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D 1H, diffusion (DOSY) and saturation-transfer difference (STD) NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site. [ABSTRACT FROM AUTHOR]

The application of thiol-yne/thiol-ene reactions to synthesize mono- and bicyclic-stapled peptides and proteins is reported. First, a thiol-ene-based peptide-stapling method in aqueous conditions was developed. This method enabled the efficient stapling of recombinantly expressed coil-coiled proteins. The resulting stapled protein demonstrated higher stability in its secondary structure than the unstapled version. Furthermore, a thiol-yne coupling was performed by using an α,ω-diyne to react with two cysteine residues to synthesize a stapled peptide with two vinyl sulfide groups. The stapled peptide could further react with another biscysteine peptide to yield a bicyclic stapled peptide with enhanced properties. For example, the cell permeability of a stapled peptide was further increased by appending an oligoarginine cell-penetrating peptide. The robustness and versatility of thiol-yne/thiol-ene reactions that can be applied to both synthetic and expressed peptides and proteins were demonstrated. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) microtubes with an interior surface composed of Pt nanoparticles (PtNPs) are self-propelled in aqueous H2O2 medium. They can capture cyanine dye and Escherichia coli ( E. coli) efficiently. Microtubes were prepared by wet templating synthesis by using a track-etched polycarbonate (PC) membrane with alternate filtrations of aqueous HSA, poly- l-arginine (PLA), and citrate-PtNPs. Subsequent dissolution of the PC template yielded uniform hollow cylinders made of (PLA/HSA)8PLA/PtNP stacking layers (1.16±0.02 μm outer diameter, ca. 23 μm length). In aqueous H2O2 media, the soft protein microtubes are self-propelled by jetting O2 bubbles from the open-end terminus. The effects of H2O2 and surfactant concentrations on the velocity were investigated. The swimming microtube captured cyanine dye in the HSA component of the wall. Addition of an intermediate γ-Fe3O4 layer allowed manipulation of the direction of movement of the tubule by using a magnetic field. Because the exterior surface is positively charged, the bubble-propelled microtubes adsorbed E. coli with high efficiency. The removal ratio of E. coli by a single treatment reached 99 %. [ABSTRACT FROM AUTHOR]

The synthesis of polymer-protein nanostructures opens up a new avenue for the development of new biomaterials. In this research, covalently connected polymer-protein nanostructures were fabricated through a reactive self-assembly approach. Poly( tert-butyl methacrylate- co-pyridyl disulfide methacrylamide) (P tBMA- co-PPDSMA) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Covalently connected nanostructures (CCNs) with hydrophobic polymer cores and hydrophilic protein coronae were prepared by adding solutions of P tBMA- co-PPDSMA/DMF to aqueous solutions of bovine serum albumin (BSA). The thiol-disulfide exchange reaction between pyridyl disulfide groups on the polymer chains and thiol groups on the protein molecules plays a key role in the fabrication of CCNs. The self-assembly process was investigated by dynamic light scattering (DLS) and stopped-flow techniques. DLS results indicated that the sizes of the CCNs were determined by the initial polymer concentration, the BSA concentration, and the average number of thiol groups on BSA molecules. TEM and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to analyze the nanostructures. Far-UV circular dichroism results demonstrated that the original folded conformations of BSA molecules were basically maintained in the reactive self-assembly process. Compared with native BSA, the secondary structure and conformation change of coronal BSA induced by urea or thermal treatment were remarkably suppressed. The cytotoxicity assays demonstrated that the CCNs were essentially nontoxic to Hela and COS-7 cells. [ABSTRACT FROM AUTHOR]

The molecular interactions between the CeIV-substituted Keggin anion [PW11O39Ce(OH2)4]3− ( CeK) and hen egg-white lysozyme (HEWL) were investigated by molecular dynamics simulations. The analysis of CeK was compared with the CeIV-substituted Keggin dimer [(PW11O39)2Ce]10− ( CeK2) and the ZrIV-substituted Lindqvist anion [W5O18Zr(OH2)(OH)]3− ( ZrL) to understand how POM features such as shape, size, charge, or type of incorporated metal ion influence the POM⋅⋅⋅protein interactions. Simulations revealed two regions of the protein in which the CeK anion interacts strongly: cationic sites formed by Arg21 and by Arg45 and Arg68. The POMs chiefly interact with the side chains of the positively charged (arginines, lysines) and the polar uncharged residues (tyrosines, serines, aspargines) via electrostatic attraction and hydrogen bonding with the oxygen atoms of the POM framework. The CeK anion shows higher protein affinity than the CeK2 and ZrL anions, because it is less hydrophilic and it has the right size and shape for establishing interactions with several residues simultaneously. The larger, more negatively charged CeK2 anion has a high solvent-accessible surface, which is sub-optimal for the interaction, while the smaller ZrL anion is highly hydrophilic and cannot efficiently interact with several residues simultaneously. [ABSTRACT FROM AUTHOR]

Front Cover: A Quadrupolar Bis-Triarylborane Chromophore as a Fluorimetric and Chirooptic Probe for Simultaneous and Selective Sensing of DNA, RNA and Proteins (Chem. Eur. J. 10/2020) The applicability of such fluorimetric differentiation in vitro was demonstrated, strongly supporting a protein-like target as a dominant binding site of this dye in human cells. The applicability of such fluorimetric differentiation in vitro was demonstrated, strongly supporting a protein-like target as a dominant binding site of this dye in human cells. [Extracted from the article]

Of the model intrinsically disordered Starmaker-like (Stm-l) protein, which is active in fish otoliths biomineralization, within calcium carbonate crystals was studied in vitro. Of the model intrinsically disordered Starmaker-like (Stm-l) protein, which is active in fish otoliths biomineralization, within calcium carbonate crystals was studied in vitro. The Stm-l promotes calcium carbonate nucleation and anisotropic tailoring of crystal morphology. [Extracted from the article]

Conformation Analysis NMR spectroscopy is a powerful method to investigate protein structure and dynamics, but backbone resonances in large proteins lead to difficulties. A covariance method utilizes the linear correlation coefficient between two degenerate 13Cα peaks to accelerate assignment; a 42 kDa MDP protein backbone was assigned with 98 % accuracy. For more details, see the Full Paper by K. Ruan, J. Wu, et al. on page 9556 ff. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) causes severe infections with only few effective antibiotic therapies currently available. To approach this challenge, chemical entities with a novel and resistance-free mode of action are desperately needed. Here, we introduce a new hydroxyamide compound that effectively reduces the expression of devastating toxins in various S. aureus and MRSA strains. The molecular mechanism was investigated by transcriptome analysis as well as by affinity-based protein profiling. Down-regulation of several pathogenesis associated genes suggested the inhibition of a central virulence-related pathway. Mass spectrometry-based chemical proteomics revealed putative molecular targets. Systemic treatment with the hydroxyamide showed significant reduction of abscess sizes in a MRSA mouse skin infection model. The absence of resistance development in vitro further underlines the finding that targeting virulence could lead to prolonged therapeutic options in comparison to antibiotics that directly address bacterial survival. [ABSTRACT FROM AUTHOR]

The interfacial properties of solid substrates are of importance for protein adsorption. Herein, we report a reversible protein adsorption switch based on the host-guest interaction of the butoxy pillar[5]arene and adipic acid. By the detector of the contact angle (CA), atomic force microscopy (AFM), and luminoscope on the silicon substrate, the intelligent protein switch exhibits excellent adsorptivity for BSA and switch performance by pH regulation. [ABSTRACT FROM AUTHOR]

The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for Nα-deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca2+ ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production. [ABSTRACT FROM AUTHOR]

Platinum anticancer drugs are particularly in need of controlled drug delivery because of their severe side effects. Platinum(IV) agents are designed as prodrugs to reduce the side effects of platinum(II) drugs; however, premature reduction could limit the effect as a prodrug. In this work, a highly biocompatible, pH and redox dual-responsive delivery system is prepared by using hybrid nanoparticles of human serum albumin (HSA) and calcium phosphate (CaP) for the PtIV prodrug of cisplatin. This conjugate is very stable under extracellular conditions, so that it protects the platinum(IV) prodrug in HSA. Upon reaching the acidic and hypoxic environment, the platinum drug is released in its active form and is able to bind to the target DNA. The Pt-HSA/CaP hybrid inhibits the proliferation of various cancer cells more efficiently than cisplatin. Different cell cycle arrests suggest different cellular responses of the PtIV prodrug in the CaP nanocarrier. Interestingly, this delivery system demonstrates enhanced cytotoxicity to tumor cells, but not to normal cells. [ABSTRACT FROM AUTHOR]

Highlights from the article: B Pyruvate formate-lyase b (PFL) catalyzes the reversible conversion of pyruvate and coenzyme A (CoA) into formate and acetyl-CoA. B Pyruvate formate-lyase b (PFL) catalyzes the reversible conversion of pyruvate and coenzyme A (CoA) into formate and acetyl-CoA. Coenzyme A, molecular dynamics, protein dynamics, pyruvate formate-lyase, radical enzymes.

A High ‐ Mannose ‐ Type Glycan Library The biological roles of protein glycosylation are well recognized. The addition of asparagine ‐ linked glycan chains is particularly important and the critical roles of high ‐ mannose ‐ type oligosaccharides in various aspects of glycoprotein quality control have attracted much attention in recent years. In their Full Paper on page 3224 ff., Y. Ito et al. report a chemo ‐ enzymatic strategy for the construction of a high ‐ mannose ‐ type glycan library by systematic chemo ‐ enzymatic trimming of a single Man9 ‐ based precursor that overcomes the limitation associated with the use of Gal residue as a capping element. The synthetic route led to a fluorescently labeled tridecasaccharide as the common precursor and the preparation of a complete set of mono ‐ and non ‐ glucosylated high ‐ mannose ‐ type glycans. The power of the methodology was demonstrated by the interaction with human recombinant N ‐ acetylglucosaminyltransferase ‐ I toward M7 ‐ M3 glycans, clarifying the glycan specificity in the context of high ‐ mannose ‐ type glycans. [ABSTRACT FROM AUTHOR]

Natural products represent a rich source of bioactive compounds that constitute a large fraction of approved drugs. Among those are molecules with electrophilic scaffolds, such as Michael acceptors, β-lactams, and epoxides that irreversibly inhibit essential enzymes based on their catalytic mechanism. In the search for novel bioactive molecules, current methods are challenged by the frequent rediscovery of known chemical entities. Herein small nucleophilic probes that attack electrophilic natural products and enhance their detection by HPLC-UV and HPLC-MS are introduced. A screen of diverse probe designs revealed one compound with a desired selectivity for epoxide- and maleimide-based antibiotics. Correspondingly, the natural products showdomycin and phosphomycin could be selectively targeted in extracts of their natural producing organism, in which the probe-modified molecules exhibited superior retention and MS detection relative to their unmodified counterparts. This method may thus help to discover small, electrophilic molecules that might otherwise easily elude detection in complex samples. [ABSTRACT FROM AUTHOR]

Vicinal-sulfydryl-containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (-CGPC-) of human thioredoxin, two active-site-matched fluorescent probes, o-Dm-Ac and m-Dm-Ac, were developed for real-time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W-6), WCGGPCK (W-7), and WCGGGPCK (W-8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o-Dm-Ac displays a higher binding sensitivity with the above-mentioned peptides and proteins, especially with W-7 and rTrx. Furthermore, o-Dm-Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe-design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ. [ABSTRACT FROM AUTHOR]

Recognition of furanosides (five-membered ring sugars) by proteins plays important roles in host-pathogen interactions. In comparison to their six-membered ring counterparts (pyranosides), detailed studies of the molecular motifs involved in the recognition of furanosides by proteins are scarce. Here the first in-depth molecular characterization of a furanoside-protein interaction system, between an antibody (CS-35) and cell wall polysaccharides of mycobacteria, including the organism responsible for tuberculosis is reported. The approach was centered on the generation of the single chain variable fragment of CS-35 and a rational library of its mutants. Investigating the interaction from various aspects revealed the structural motifs that govern the interaction, as well as the relative contribution of molecular forces involved in the recognition. The specificity of the recognition was shown to originate mainly from multiple CH-π interactions and, to a lesser degree, hydrogen bonds formed in critical distances and geometries. [ABSTRACT FROM AUTHOR]

This work reports a novel chlorooxime mediated modification of native peptides and proteins under physiologic conditions. This method features fast reaction kinetics (apparent k2=306±4 M−1s−1 for GSH) and exquisite selectivity for cysteine residues. This cysteine conjugation reaction can be carried out with just single‐digit micromolar concentrations of the labeling reagent. The conjugates show high stability towards acid, base, and external thiol nucleophiles. A nitrile oxide species generated in situ is likely involved as the key intermediate. Furthermore, a bis‐chlorooxime reagent is synthesized to enable facile Cys‐Cys stapling in native peptides and proteins. This highly efficient cysteine conjugation and stapling was further implemented on bacteriophage to construct chemically modified phage libraries. [ABSTRACT FROM AUTHOR]

Site specific installation of a paramagnetic ion with magnetic anisotropy in a biomolecule generates valuable structural restraints, such as pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs). These paramagnetic effects can be used to characterize the structures, interactions and dynamics of biological macromolecules and their complexes. Two single‐armed DOTA‐like tags, BrPSPy‐DO3M(S)A‐Ln and BrPSPy‐6M‐DO3M(S)A‐Ln, each containing a thiol‐specific reacting group, that is, a phenylsulfonyl pyridine moiety, are demonstrated as rigid, reactive and stable paramagnetic tags for protein modification by formation of a reducing resistant thioether bond between the protein and the tag. The two tags present high reactivity with the solvent exposed thiol group in aqueous solution at room temperature. The introduction of Br at the meta‐position in pyridine enhances the reactivity of 4‐phenylsulfonyl pyridine towards the solvent exposed thiol group in a protein, whereas the ortho‐methyl group in pyridine increases the rigidity of the tag in the protein conjugates. The high performance of these two tags has been demonstrated in different cysteine mutants of ubiquitin and GB1. The high reactivity and rigidity of these two tags can be added in the toolbox of paramagnetic tags suitable for the high‐resolution NMR measurements of biological macromolecules and their complexes. [ABSTRACT FROM AUTHOR]

A selection of bioactive polyphenols of different structural classes, such as the ellagitannins vescalagin and vescalin, the flavanoids catechin, epicatechin, epigallocatechin gallate (EGCG), and procyanidin B2, and the stilbenoids resveratrol and piceatannol, were chemically modified to bear a biotin unit for enabling their immobilization on streptavidin‐coated sensor chips. These sensor chips were used to evaluate in real time by surface plasmon resonance (SPR) the interactions of three different surface‐bound polyphenolic ligands per sensor chip with various protein analytes, including human DNA topoisomerase IIα, flavonoid leucoanthocyanidin dioxygenase, B‐cell lymphoma 2 apoptosis regulator protein, and bovine serum albumin. The types and levels of SPR responses unveiled major differences in the association, or lack thereof, and dissociation between a given protein analyte and different polyphenolic ligands. Thus, this multi‐analysis SPR technique is a valuable methodology to rapidly screen and qualitatively compare various polyphenol–protein interactions. [ABSTRACT FROM AUTHOR]

Drug delivery vectors based on amphiphiles have important features such as versatile physicochemical properties and stimuli‐responsiveness. Amino acid‐based surfactants are especially promising amphiphiles due to their enhanced biocompatibility compared to conventional surfactants. They can self‐organize into micelles, vesicles and complex hierarchical structures, such as fibers, twisted and coiled ribbons, and tubules. In this work, we investigated the self‐assembly and drug loading properties of a family of novel anionic double‐tailed lysine‐derived surfactants, with variable degree of tail length mismatch, designated as mLys10 and 10Lysn, where m and n are the number of carbon atoms in the tails. These surfactants form tubular aggregates with assorted morphologies in water that undergo gelation due to dense entanglement, as evidenced by light and electron microscopy. Lysozyme (LZM), an enzyme with antimicrobial properties, was selected as model protein for loading. After the characterization of the interfacial properties and phase behavior of the amphiphiles, the LZM‐loading ability of the tubules was investigated, under varying experimental conditions, to assess the efficiency of the aggregates as pH‐ and temperature‐sensitive nanocarriers. Further, the toxicological profile of the surfactants per se and surfactant/LZM hydrogels was obtained, using human skin fibroblasts (BJ‐5ta cell line). Overall, the results show that the tubule‐based hydrogels exhibit very interesting properties for the transport and controlled release of molecules of therapeutic interest. [ABSTRACT FROM AUTHOR]

Over the last two decades, the development of chemical biology and the need for more defined protein conjugates have fostered active research on new bioconjugation techniques. In particular, a wide range of biorthogonal labelling strategies have been reported to functionalise the phenol side chain of tyrosines (Tyr). Tyr occur at medium frequency and are partially buried at the protein surface, offering interesting opportunities for site‐selective labelling of the most reactive residues. Tyr‐targeting has proved effective for designing a wide range of important biomolecules including antibody–drug conjugates, fluorescent or radioactive protein probes, glycovaccines, protein aggregates, and PEG conjugates. Innovative methods have also been reported for site‐specific labelling with ligand‐directed anchors and for the specific affinity capture of proteins. This review will present and discuss these promising alternatives to the conventional labelling of the nucleophilic lysine and cysteine residues. [ABSTRACT FROM AUTHOR]

Frontispiece: Structure-Activity Relationships for the Affinity of Chaotropic Polyoxometalate Anions towards Proteins Keywords: chaotropic anions; molecular dynamics; polyoxometalates; protein interactions; structure-activity relationships EN chaotropic anions molecular dynamics polyoxometalates protein interactions structure-activity relationships 1 1 1 05/09/20 20200507 NES 200507 B Atomistic molecular dynamics b simulations and QSAR-like modeling techniques were applied to establish relationships between the structure of chaotropic polyoxometalate (POM) anions and their affinity towards biological systems. Chaotropic anions, molecular dynamics, polyoxometalates, protein interactions, structure-activity relationships. [Extracted from the article]

Invited for the cover of this issue are the groups of Todd B. Marder at the Julius‐Maximilians‐Universität Würzburg and Ivo Piantanida at the Ruder Boškovic Institute. The image depicts the molecular structure of a bis‐triarylborane‐based chromophore that is simultaneously detecting ds‐DNA and proteins. Read the full text of the article at10.1002/chem.201903936. [ABSTRACT FROM AUTHOR]

Cover Feature: Reductant-Induced Free Radical Fluoroalkylation of Nitrogen Heterocycles and Innate Aromatic Amino Acid Residues in Peptides and Proteins (Chem. Eur. J. 69/2019) The follow-up click reaction by using the pink reagent carrying a payload delivers the conjugate of choice. Realistic depiction of hypervalent iodine reagents includes the T-shape geometry on the iodine atom and the azido-containing fluoroalkyl group. [Extracted from the article]

Keywords: actinides; molecular speciation; proteins; radiochemistry; toxicology A relevant model was designed for the interaction of actinyls with phosphorylated proteins, which have an important role as an iron, calcium, and magnesium storage protein in egg yolk. Actinides, molecular speciation, proteins, radiochemistry, toxicology. [Extracted from the article]

A bis‐triarylborane tetracation (4‐Ar2B‐3,5‐Me2C6H2)‐C≡C−C≡C‐(3,5‐Me2C6H2‐4‐BAr2 [Ar=(2,6‐Me2‐4‐NMe3‐C6H2)+] (24+) shows distinctly different behaviour in its fluorimetric response than that of our recently published bis‐triarylborane 5‐(4‐Ar2B‐3,5‐Me2C6H2)‐2,2′‐(C4H2S)2–5′‐(3,5‐Me2C6H2‐4‐BAr2) (34+). Single‐crystal X‐ray diffraction data on the neutral bis‐triarylborane precursor 2 N confirm its rod‐like dumbbell structure, which is shown to be important for DNA/RNA targeting and also for BSA protein binding. Fluorimetric titrations with DNA/RNA/BSA revealed the very strong affinity of 24+ and indicated the importance of the properties of the linker connecting the two triarylboranes. Using the butadiyne rather than a bithiophene linker resulted in an opposite emission effect (quenching vs. enhancement), and 24+ bound to BSA 100 times stronger than 34+. Moreover, 24+ interacted strongly with ss‐RNA, and circular dichroism (CD) results suggest ss‐RNA chain‐wrapping around the rod‐like bis‐triarylborane dumbbell structure like a thread around a spindle, a very unusual mode of binding of ss‐RNA with small molecules. Furthermore, 24+ yielded strong Raman/SERS signals, allowing DNA or protein detection at ca. 10 nm concentrations. The above observations, combined with low cytotoxicity, efficient human cell uptake and organelle‐selective accumulation make such compounds intriguing novel lead structures for bio‐oriented, dual fluorescence/Raman‐based applications. [ABSTRACT FROM AUTHOR]

Metal–organic frameworks (MOFs) hold great promise for numerous applications. However, proteins, carriers of biological functions in living systems, have not yet been fully explored as building blocks for the construction of MOFs. This work presents a strategy for the fabrication of binary MOFs. Considering octahedral ferritin symmetry, four His2 (His–His) motifs were first incorporated into the exterior surface of a ferritin nanocage near each C4 channel, yielding protein linkers with multiple metal‐binding sites (bisH‐SF). Secondly, by adding nickel ions to bisH‐SF solutions triggers the self‐assembly of ferritin nanocages into a porous 3D crystalline MOF with designed protein lattice, where two adjacent ferritin molecules along the C4 symmetry axes are bridged by four dinuclear or tetranuclear nickel clusters depending on Ni2+ concentration. This work provides a simple approach for precise control over a binary protein–metal crystalline framework, and the resulting MOFs exhibited inherent ferroxidase activity and peroxidase‐like catalytic activity. [ABSTRACT FROM AUTHOR]

Pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS) offers several methods for measuring dipolar coupling constants and thus the distance between electron spin centers. Up to now, PDS measurements have been mostly applied to spin centers whose g‐anisotropies are moderate and therefore have a negligible effect on the dipolar coupling constants. In contrast, spin centers with large g‐anisotropy yield dipolar coupling constants that depend on the g‐values. In this case, the usual methods of extracting distances from the raw PDS data cannot be applied. Here, the effect of the g‐anisotropy on PDS data is studied in detail on the example of the low‐spin Fe3+ ion. First, this effect is described theoretically, using the work of Bedilo and Maryasov (Appl. Magn. Reson. 2006, 30, 683–702) as a basis. Then, two known Fe3+/nitroxide compounds and one new Fe3+/trityl compound were synthesized and PDS measurements were carried out on them using a method called relaxation induced dipolar modulation enhancement (RIDME). Based on the theoretical results, a RIDME data analysis procedure was developed, which facilitated the extraction of the inter‐spin distance and the orientation of the inter‐spin vector relative to the Fe3+g‐tensor frame from the RIDME data. The accuracy of the determined distances and orientations was confirmed by comparison with MD simulations. This method can thus be applied to the highly relevant class of metalloproteins with, for example, low‐spin Fe3+ ions. [ABSTRACT FROM AUTHOR]

Ruthenium–arene complexes are a unique class of organometallic compounds that have been shown to have prominent therapeutic potencies. Here, we have investigated the interactions of Ru‐cymene complexes with a zinc‐finger protein NCp7, aiming to understand the effects of various ligands on the reaction. Five different binding modes were observed on selected Ru‐complexes. Ru‐cymene complex can bind to proteins through either noncovalent binding alone or through a combination of covalent and noncovalent binding modes. Moreover, the noncovalent interaction can promote the coordination of RuII to NCp7, resulting synergistic effects of the different ligands. The binding of Ru(Cym) complexes leads to dysfunction of NCp7 through zinc‐ejection and structural perturbation. These results indicate that the reactivity of Ru‐complexes can be modulated by ligands through different approaches, which could be closely correlated to their different therapeutic effects. [ABSTRACT FROM AUTHOR]

The biological mediation of mineral formation (biomineralization) is realized through diverse organic macromolecules that guide this process in a spatial and temporal manner. Although the role of these molecules in biomineralization is being gradually revealed, the molecular basis of their regulatory function is still poorly understood. In this study, the incorporation and distribution of the model intrinsically disordered starmaker‐like (Stm‐l) protein, which is active in fish otoliths biomineralization, within calcium carbonate crystals, is revealed. Stm‐l promotes crystal nucleation and anisotropic tailoring of crystal morphology. Intracrystalline incorporation of Stm‐l protein unexpectedly results in shrinkage (and not expansion, as commonly described in biomineral and bioinspired crystals) of the crystal lattice volume, which is described herein, for the first time, for bioinspired mineralization. A ring pattern was observed in crystals grown for 48 h; this was composed of a protein‐enriched region flanked by protein‐depleted regions. It can be explained as a result of the Ostwald‐like ripening process and intrinsic properties of Stm‐l, and bears some analogy to the daily growth layers of the otolith. [ABSTRACT FROM AUTHOR]

Urease uses a cluster of two NiII ions to activate a water molecule for urea hydrolysis. The key to this unsurpassed enzyme is a change in the conformation of a flexible structural motif, the mobile flap, which must be able to move from an open to a closed conformation to stabilize the chelating interaction of urea with the NiII cluster. This conformational change brings the imidazole side chain functionality of a critical histidine residue, αHis323, in close proximity to the site that holds the transition state structure of the reaction, facilitating its evolution to the products. Herein, we describe the influence of the solution pH in modulating the conformation of the mobile flap. High‐resolution crystal structures of urease inhibited in the presence of N‐(n‐butyl)phosphoric triamide (NBPTO) at pH 6.5 and pH 7.5 are described and compared to the analogous structure obtained at pH 7.0. The kinetics of urease in the absence and presence of NBPTO are investigated by a calorimetric assay in the pH 6.0–8.0 range. The results indicate that pH modulates the protonation state of αHis323, which was revealed to have pKa=6.6, and consequently the conformation of the mobile flap. Two additional residues (αAsp224 and αArg339) are shown to be key factors for the conformational change. The role of pH in modulating the catalysis of urea hydrolysis is clarified through the molecular and structural details of the interplay between protein conformation and solution acidity in the paradigmatic case of a metalloenzyme. [ABSTRACT FROM AUTHOR]

A simple and efficient strategy for the selective modification of the peptide N terminus with an unnatural amino acid is described. A peptide having a SUMO‐HisTag‐TEV sequence (SUMO: small ubiquitin‐related modifier, TEV: tobacco etch virus) preceding the N terminus of the target peptide was designed. Recombinant expression in E. coli and subsequent SUMO protease cleavage yielded the HisTag‐TEV‐target peptide. Partial protection of the lysine side chains of this peptide with d‐glucopyranosyloxycarbonyl and removal of the HisTag‐TEV sequence by TEV protease yielded the partially protected peptide with a free N‐terminal amine. Coupling of selenocysteine selectively at the N terminus and subsequent acidic deprotection of the carbohydrate protecting groups yielded a modified peptide that can be used for native chemical ligation (NCL). As a proof of concept, the modification of a longer recombinant peptide with selenocysteinylserine (GalNAc) at the N terminus was demonstrated. [ABSTRACT FROM AUTHOR]

Synthesizing proteins inside liposomes and other microcompartments is a well‐established practice. However, the origin of this research is not from the distant past, dating back to 1999–2004, when the first successful attempts were published. Protein synthesis inside artificial compartments is now under strong expansion in the context of synthetic biology (in bottom‐up approaches), and, in particular, it strongly contributes to the construction of artificial cell‐like systems. These systems, often called "synthetic cells", can be used to model cellular processes, including membrane‐centered ones. They are very innovative models that complement traditional studies and promise future applications. This review does not discuss all current directions in synthetic cell research; in particular, it does not include all kinds of artificial compartments. Instead, it is uniquely dedicated to the analysis of historical and technical developments of protein synthesis inside liposomes, highlighting a selected list of open questions. One of the goals is to note the importance of mastering liposome technology together with cell‐free systems for the successful realization of this specific type of synthetic cell. With this aim, four currently employed protocols are compared and discussed, with a major emphasis on the droplet transfer method, which is attractive due to its simplicity and encapsulation efficiency. [ABSTRACT FROM AUTHOR]

Ligand‐based control of protein functional motions can provide novel opportunities in the study of fundamental biological mechanisms and in the development of novel therapeutics. In this work we addressed the ligand‐based modulation of integrin functions. Inhibitors of integrin αvβ3 are interesting anticancer agents but their molecular mechanisms are still unclear: Peptides and peptidomimetics characterized by the Arg‐Gly‐Asp (RGD) or isoAsp‐Gly‐Arg (isoDGR) binding motifs have shown controversial agonist/antagonist effects. We have investigated the differential mechanisms of integrin activation/deactivation by three distinct ligands (cyclo‐RGDf(NMe)V (Cilengitide), cyclo[DKP3‐RGD], cyclo[DKP3‐isoDGR]; DKP=diketopiperazine) through a comparative analysis of ligand‐controlled protein internal dynamics: Although RGD facilitates the onset of dynamic states leading to activation, isoDGR induces a diffuse rigidification of the complex consistent with antagonist activities. Computational predictions have been experimentally probed by showing that the antibody AP5, which is capable of recognizing the active form of integrin, binds specifically to the RGD complexes and not to the isoDGR complex, which supports opposite functional roles of the two motifs targeting the same binding site. [ABSTRACT FROM AUTHOR]