Visualizing the Adenylation Activities and Protein–Protein Interactions of Aryl Acid Adenylating Enzymes

Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, as they serve as covalent tethers for amino acid and aryl acid substrates and enable the growth of peptide intermediates. Strategies are therefore required to study protein–protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein–protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of the aryl acid A domains. If coupled with in-gel fluorescence imaging, this strategy allows visualization of the protein–protein interactions required to recognize and transfer the substrate to the partner ArCP. This technique has potential for the analysis of protein–protein interactions within these biosynthetic enzymes at the molecular level and for use in the combinatorial biosynthesis of new nonribosomal peptides.     

Three-Dimensional Structural Aspects of Protein–Polysaccharide Interactions

Linear polysaccharides are typically composed of repeating mono- or disaccharide units and are ubiquitous among living organisms. Polysaccharide diversity arises from chain-length variation, branching, and additional modifications. Structural diversity is associated with various physiological functions, which are often regulated by cognate polysaccharide-binding proteins. Proteins that interact with linear polysaccharides have been identified or developed, such as galectins and polysaccharide-specific antibodies, respectively. Currently, data is accumulating on the three-dimensional structure of polysaccharide-binding proteins. These proteins are classified into two types: exo-type and endo-type. The former group specifically interacts with the terminal units of polysaccharides, whereas the latter with internal units. In this review, we describe the structural aspects of exo-type and endo-type protein-polysaccharide interactions. Further, we discuss the structural basis for affinity and specificity enhancement in the face of inherently weak binding interactions.     

Targeted Nanoswitchable Inhibitors of Protein–Protein Interactions Involved in Apoptosis

Progress in drug delivery is hampered by a lack of efficient strategies to target drugs with high specificity and precise spatiotemporal regulation. The remote control of nanoparticles and drugs with light allows regulation of their action site and dosage. Peptide-based drugs are highly specific, non-immunogenic, and can be designed to cross the plasma membrane. In order to combine target specificity and remote control of drug action, here we describe a versatile strategy based on a generalized template to design nanoswitchable peptides that modulate protein–protein interactions upon light activation. This approach is demonstrated to promote photomodulation of two important targets involved in apoptosis (the interactions Bcl-xL–Bak and MDM2–p53), but can be also applied to a large pool of therapeutically relevant protein–protein interactions mediated by α-helical motifs. The template can be adjusted using readily available information about hot spots (residues contributing most to the binding energy) at the protein–protein interface of interest.     

Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein–Protein Interactions of BRCA1 tBRCT Domain

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure–activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein–protein interactions.     

Multivalent Ligands with Tailor-Made Anion Binding Motif as Stabilizers of Protein–Protein Interactions

Modulation of protein–protein interactions (PPIs) is essential for understanding and tuning biologically relevant processes. Although inhibitors for PPIs are widely used, the field still lacks the targeted design of stabilizers. Here, we report unnatural stabilizers based on the combination of multivalency effects and the artificial building block guanidiniocarbonylpyrrol (GCP), an arginine mimetic. Unlike other GCP-based ligands that modulate PPIs in different protein targets, only a tetrameric design shows potent activity as stabilizer of the 14-3-3ζ/C-Raf and 14-3-3ζ/Tau complexes in the low-micromolar range. This evidences the role of multivalency for achieving higher specificity in the modulation of PPIs.     

Modulating Protein–Protein Interactions with Visible-Light-Responsive Peptide Backbone Photoswitches

Life relies on a myriad of carefully orchestrated processes, in which proteins and their direct interplay ultimately determine cellular function and disease. Modulation of this complex crosstalk has recently attracted attention, even as a novel therapeutic strategy. Herein, we describe the synthesis and characterization of two visible-light-responsive peptide backbone photoswitches based on azobenzene derivatives, to exert optical control over protein–protein interactions (PPI). The novel peptidomimetics undergo fast and reversible isomerization with low photochemical fatigue under alternatively blue-/green-light irradiation cycles. Both bind in the nanomolar range to the protein of interest. Importantly, the best peptidomimetic displays a clear difference between isomers in its protein-binding capacity and, in turn, in its potential to inhibit enzymatic activity through PPI disruption. In addition, crystal structure determination, docking and molecular dynamics calculations allow a molecular interpretation and open up new avenues in the design and synthesis of future photoswitchable PPI modulators.     

Analysis of Protein–Protein Interactions in MCF-7 and MDA-MB-231 Cell Lines Using Phthalic Acid Chemical Probes

Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein–protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively.     

Discovery of Small-Molecule Modulators of Protein–RNA Interactions by Fluorescence Intensity-Based Binding Assay

Protein–RNA interactions mediate various cellular processes, the dysregulation of which has been associated with a list of diseases. Thus, novel experimental tools for monitoring protein–RNA interactions are highly desirable to identify new chemical modulators of these therapeutic targets. In this study, we constructed simple fluorescence intensity-based protein–RNA binding assays by testing multiple environment-sensitive organic fluorophores. We selected the oncogenic interaction between Lin28 and the let-7 microRNA and the important immunomodulatory Roquin–Tnf CDE interaction as representative targets. We adapted this assay to high-throughput screening for the identification of pyrazolyl thiazolidinedione-type molecules as potent small-molecule inhibitors of protein–microRNA interactions. We clearly showed the structure–activity relationships of this new class of Lin28–let-7 interaction inhibitors, and confirmed that cellular mature let-7 microRNAs and their target genes could be modulated upon treatment with the pyrazolyl thiazolidinedione-type inhibitor. We expect that our simple and adaptable screening approach can be applied for the development of various assay systems aimed at the identification of bioactive small molecules targeting protein–RNA interactions.     

Novel Dextran-Supported Biological Probes Decorated with Disaccharide Entities for Investigating the Carbohydrate–Protein Interactions of Gal-3

The quest for novel natural-like biomolecular probes that can be used to gain information on biological recognition events is of topical interest to several scientific areas. In particular, the recognition of carbohydrates by proteins modulates a number of important biological processes. These molecular recognition events are, however, difficult to study by the use of naturally occurring oligosaccharides and polysaccharides owing to their intrinsic structural heterogeneity and to the many technical difficulties encountered during the isolation of sufficient quantities of pure material for detailed structural and biological studies. Therefore, the construction of homogenous biomolecular probes that can mimic both the biophysical properties of polysaccharide backbones and the properties of bioactive oligosaccharide fragments are highly sought after. Herein, synthetic methodology for the construction of well-defined bioconjugates consisting of biologically relevant disaccharide fragments grafted onto a dextran backbone is presented, and a preliminary NMR spectroscopy study of their interactions with galectin-3 as a model lectin is conducted.     

Modeling,Simulation, and Experimental Validation of Drying and Denaturation Behavior of Whey Protein Isolate–Based Coatings

Previous studies proved that heat-treated whey protein isolate (WPI)-based coatings can act as an adequate packaging material with sufficient barrier properties and producibility. These properties also depend on the degree of denaturation of the coating formulations used. This study focuses on the modeling, simulation, and experimental validation of drying and denaturation behavior of WPI-based coatings. Simulation according to existing models is able to describe denaturation and drying kinetics of WPI based coatings adequately. A degree of denaturation of about 13% was reached during coating production. Water vapor transmission rates were reduced up to 67%.     

Microstructure, Properties and Lignin-Based Modification of Wood–Ceramics from Rice Husk and Coal Tar Pitch

Wood–Ceramics are prepared from a shaped mixture of rice husk and high-temperature coal tar pitch by pyrolysis at different temperatures. Subsequently, the wood–ceramics are modified by pyrolysis of different mixture precursors with adding lignin. The effect of pyrolysis temperature, lignin content and lignin types on the microstructure, yield, volume shrinkage, open porosity ratio and bending strength of the wood–ceramics is discussed, respectively. The results show that the microstructure, volume shrinkage ratio, and bending strength of wood–ceramics are significantly dependent on the pyrolysis temperature, lignin content and types. When the pyrolysis temperature increases from 500 to 1200 °C, the (002) crystal planes peaks of the wood–ceramics move to a higher diffraction angle and the intensity of these peaks increase. The volume shrinkage ratio and bending strength of the wood–ceramics increase from 41.2 to 55.5 % and 10.2 to 31.2 MPa, respectively. Moreover, the open porosity ratio first decreases from 35.6 to 26.2 %, and then increases to 30.1 %. The enzymatic hydrolysis lignin (EHL) plays an important role in improving the order and graphitization, porous content and open porosity of the EHL-modified wood–ceramics, whereas the lignosulfonate causes the bending strength of the lignosulfonate-modified wood–ceramics to decrease.     

Design,Synthesis, and Self-Assembly Studies of a Suite of Monodisperse,Facially Amphiphilic,Protein–Dendron Conjugates

The custom design of protein–dendron amphiphilic macromolecules is at the forefront of macromolecular engineering. Macromolecules with this architecture are very interesting because of their ability to self-assemble into various biomimetic nanoscopic structures. However, to date, there are no reports on this concept due to technical challenges associated with the chemical synthesis. Towards that end, herein, a new chemical methodology for the modular synthesis of a suite of monodisperse, facially amphiphilic, protein–dendron bioconjugates is reported. Benzyl ether dendrons of different generations (G1–G4) are coupled to monodisperse cetyl ethylene glycol to form macromolecular amphiphilic activity-based probes (AABPs) with a single protein reactive functionality. Micelle-assisted protein labeling technology is utilized for site-specific conjugation of macromolecular AABPs to globular proteins to make monodisperse, facially amphiphilic, protein–dendron bioconjugates. These biohybrid conjugates have the ability to self-assemble into supramolecular protein nanoassemblies. Self-assembly is primarily mediated by strong hydrophobic interactions of the benzyl ether dendron domain. The size, surface charge, and oligomeric state of protein nanoassemblies could be systematically tuned by choosing an appropriate dendron or protein of interest. This chemical method discloses a new way to custom-make monodisperse, facially amphiphilic, protein–dendron bioconjugates.     

Atomic Force Microscopy Study of Protein–Protein Interactions in the Cytochrome CYP11A1 (P450scc)-Containing Steroid Hydroxylase System

Atomic force microscopy (AFM) and photon correlation spectroscopy (PCS) were used for monitoring of the procedure for cytochrome CYP11A1 monomerization in solution without phospholipids. It was shown that the incubation of 100 μM CYP11A1 with 12% Emulgen 913 in 50 mM KP, pH 7.4, for 10 min at T = 22°C leads to dissociation of hemoprotein aggregates to monomers with the monomerization degree of (82 ± 4)%. Following the monomerization procedure, CYP11A1 remained functionally active. AFM was employed to detect and visualize the isolated proteins as well as complexes formed between the components of the cytochrome CYP11A1-dependent steroid hydroxylase system. Both Ad and AdR were present in solution as monomers. The typical heights of the monomeric AdR, Ad and CYP11A1 images were measured by AFM and were found to correspond to the sizes 1.6 ± 0.2 nm, 1.0 ± 0.2 nm and 1.8 ± 0.2 nm, respectively. The binary Ad/AdR and AdR/CYP11A1_mon complexes with the heights 2.2 ± 0.2 nm and 2.8 ± 0.2 nm, respectively, were registered by use of AFM. The Ad/CYP11A1_mon complex formation reaction was kinetically characterized based on optical biosensor data. In addition, the ternary AdR/Ad/CYP11A1 complexes with a typical height of 4 ± 1 nm were AFM registered.     

Intrinsic Fluorescence Excitation–Emission Matrix Spectral Features of Cottonseed Protein Fractions and the Effects of Denaturants

To better understand the functional and physicochemical properties of cottonseed protein, we investigated the intrinsic fluorescence excitation–emission matrix (EEM) spectral features of cottonseed protein isolate (CSPI) and sequentially extracted water (CSPw) and alkali (CSPa) protein fractions, and the effects of denaturants urea, guanidine hydrochloride, and sodium dodecyl sulfate. The EEM showed two contour peaks at the excitation wavelengths of 226 nm (Peak 1) and 277 nm (Peak 2). Addition of denaturants gradually shifted the emission maxima of both peaks from 335 nm to around 353 nm for CSPI and CSPa. The emission maximum (353 nm) of CSPw was unchanged by denaturation. These observations indicated that the tryptophan residues (fluorescence source) in the native CSPI and CSPa were protected within the micro hydrophobic environment, and gradually become water accessible with progressing denaturation. On the other hand, the tryptophan residues in native CSPw were already in contact with water. However, the fluorescence intensity of Peak 1 of all three protein samples decreased with increasing denaturant concentrations, suggesting similarity in some conformational changes in the three samples. Further exploration of the fluorescence mechanism of Peak 1 is needed to understand such similar conformational changes.     

Soaking Conditions Affect the Contents of Tocopherols,Tocotrienols, and γ-Oryzanol in Pigmented and Non-Pigmented Brown Rice

Tocopherols (Toc), tocotrienols (T3), and γ‐oryzanol (GO), important lipid antioxidants in rice, are known to possess various pharmacological properties. In this study, our aim was to examine the effects of soaking conditions on the extractable contents of Toc, T3, and GO in cooked pigmented (PBR) and non‐pigmented brown rice (non‐PBR). The results showed that Toc, T3, and GO concentrations in non‐PBR peaked after soaking at 25 °C for 120 min, whereas soaking conditions showed minimal effects on these compounds in PBR. In non‐PBR, high Toc, T3, and GO levels were also noted in the treatments at higher soaking temperatures (45 and 55 °C) with a shorter soaking time (less than 60 min). The GO level in both cultivars was at least ten times greater than the total vitamin E content. Vitamin E analogues having concentrations greater than 10 mg/kg in PBR and non‐PBR were γ‐Toc and γ‐T3, and α‐Toc and γ‐T3, respectively. This study concludes that presoaking and cooking processes can improve the extractable contents of Toc, T3, and GO in cooked PBR and non‐PBR, and the extraction yield of these bioactive compounds was significantly different between cultivars.     

Preparation and Surface Activities of Modified Soy Protein–Dextrin Surfactants

A series of polymeric surfactants has been prepared through the reaction of soy protein with polyethoxylated stearyl ethers of various hydrophilic chain lengths. These surfactants exhibited surface activity, evaluated using surface tension, foaming, and wetting power that was superior to that of traditional surfactants containing only one hydrophobic moiety and one hydrophilic head group. Changing the ethoxylate (EO) group length had a significant effect on the surface activity. Increasing the EO group length decreased the critical micelle concentration (CMC) and increased the surface tension at the CMC (γ_CMC). The good surface properties of these polysaccharide/protein‐type surfactants suggest that they could be used as emulsifiers to prepare oil‐in‐water emulsions displaying good stability.     

Development and Application of a Label-Free Fluorescence Method for Determining the Composition of Gold Nanoparticle–Protein Conjugates

A method was developed for determining the composition of the conjugates between gold nanoparticles and proteins based on the intrinsic fluorescence of unbound protein molecules. The fluorescence was evaluated after separation of the conjugates from the reaction mixture by centrifugation. Gold nanoparticles obtained using the citrate technique (average diameter 24 nm) were conjugated at pH 5.4 with the following four proteins: human immunoglobulin G (IgG), bovine serum albumin (BSA), recombinant streptococcal protein G (protein G), and Kunitz-type soybean trypsin inhibitor (STI). The compositions of these conjugates were determined using the developed method. The conjugate compositions were dependent on the concentration of the added protein, and in all cases reached saturation. The equilibrium dissociation constants of the gold nanoparticle conjugates with IgG, BSA, protein G, STI in the initial section of the concentration dependence curve were 4, 6, 10, and 15 nM, respectively. Close to saturation, the corresponding values were 25, 76, 175, and 100 nM, respectively. The maximal binding capacities of a single gold nanoparticle for IgG, BSA, Protein G, and STI were 52, 90, 500, and 550, respectively, which agrees well with the hypothesis of monolayer immobilization.     

The Interactions between Spheres and between a Sphere and a Half-Space,Based on the Lennard–Jones Potential

This paper investigates the interaction between two rigid spheres and between a rigid sphere and a rigid half-space, based on the Lennard–Jones potential. By using the divergence theorem and integrating the Lennard–Jones potential over the surfaces, the analytical forms of the surface tractions were obtained, and by integrating the Lennard–Jones potential over the volumes, the analytical form of the total force between two rigid spheres was obtained. The results are compared to those with the Derjaguin approximations and with the parabolic approximation for the sphere profile. The accuracy of the Derjaguin approximation and the parabolic approximation were estimated. The analytical surface traction can be used for the adhesive contact between an elastic body and a rigid one.     

Effect of Processing Time on the Tensile,Morphological, and Thermal Properties of Rice Husk Powder‐Filled Polypropylene Composites

Abstract

Brabender plasticorder internal mixer was used to incorporate rice husk powder into polypropylene. Rice husk powder (RHP)‐filled polypropylene (PP) composite was prepared by varying mixing time under constant rotor speed (50 rpm) at two mixing temperatures. Brabender mixing torque, tensile properties, morphology, and thermal properties were investigated in detail. Results indicate that the tensile properties, such as Young's modulus, yield strength, and elongation at break, do not exhibit a significant trend with the increasing of processing time. Scanning electron microscope (SEM) micrographs reveal the better filler dispersion at longer processing time. Differential scanning calorimetry (DSC) analysis indicates nucleating ability of RHP and reduction of degree of crystallinity of PP at higher temperature and longer processing time. In fact, RHP changes the melting point of PP in the composites.