The photoreaction of the photoactive yellow protein domain in the light sensor histidine kinase Ppr is influenced by the C-terminal domains

To study the role of the C-terminal domains in the photocycle of a light sensor histidine kinase (Ppr) having a photoactive yellow protein (PYP) domain as the photosensor domain, we analyzed the photocycles of the PYP domain of Ppr (Ppr-PYP) and full-length Ppr. The gene fragment for Ppr-PYP was expressed in Escherichia coli, and it was chemically reconstituted with p-coumaric acid; the full-length gene of Ppr was coexpressed with tyrosine ammonia-lyase and p-coumaric acid ligase for biosynthesis in cells. The light/dark difference spectra of Ppr-PYP were pH sensitive. They were represented as a linear combination of two independent difference spectra analogous to the PYP(L)/dark and PYP(M)/dark difference spectra of PYP from Halorhodospira halophila, suggesting that the pH dependence of the difference spectra is explained by the equilibrium shift between the PYP(L)- and PYP(M)-like intermediates. The light/dark difference spectrum of Ppr showed the equilibrium shift toward PYP(L) compared with that of Ppr-PYP. Kinetic measurements of the photocycles of Ppr and Ppr-PYP revealed that the C-terminal domains accelerate the recovery of the dark state. These observations suggest an interaction between the C-terminal domains and the PYP domain during the photocycle, by which light signals captured by the PYP domain are transferred to the C-terminal domains.     

Chemical synthesis of lymphotactin: a glycosylated chemokine with a C-terminal mucin-like domain

The synthesis of a 93-residue chemokine, lymphotactin, containing eight sites of O-linked glycosylation, was achieved using the technique of native chemical ligation. A single GalNAc residue was incorporated at each glycosylation site using standard Fmoc-chemistry to achieve the first total synthesis of a mucin-type glycoprotein. Using this approach quantities of homogeneous material were obtained for structural and functional analysis.     

Synthesis and structural characterization of metallated bioconjugates: C-terminal labeling of amino acids with aminoferrocene

Aminoferrocene, H₂N-Fc, has been substituted to the C-terminus of six amino acids using the HBTU/HOBt coupling protocol. The synthesized bioconjugates Boc-Aaa-NH-Fc, Aaa = Gly (1), Leu (2), Phe (3), Val (4), Cys(Acm) (5), Tyr(^tBu) (6) (Acm = acetamidomethyl, ^tBu = tert-butyl), have been characterized by ¹H NMR, ¹³C NMR, EI-MS, EI-HRMS, UV and CD spectroscopies. In addition, a VT NMR study on 4 and the X-ray structure of 1 are presented.     

The biotin enzyme family: conserved structural motifs and domain rearrangements

The biotin carboxylase family is comprised of a group of enzymes that utilize a covalently bound prosthetic group, biotin, as a cofactor. These enzymes, which include acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, geranoyl-CoA carboxylase, oxaloacetate decarboxylase, methylmalonyl-CoA decarboxylase, transcarboxylase and urea amidolyase, are found in diverse biosynthetic pathways in both pro-karyotes and eukaryotes. The reactions catalyzed by most members of this group of enzymes share two common features: (1) carboxylation of biotin, apparently via the formation of a carboxyphosphate intermediate, followed by (2) transcarboxylation of CO(2) from biotin to specific acceptor molecules to yield different products. Structural determinations by NMR and X-ray crystallography, complemented by mutagenesis studies, have identified some motifs that are structurally or catalytically important. Analysis of the amino acid sequences of a number of biotin carboxylases not only shows remarkable similarities within certain domains but also that there appears to have been domain rearrangements between groups of carboxylases. Acyl-coenzyme A derivatives, which bind either as substrates or as allosteric regulators of the biotin carboxylases, do not appear to share any of the CoA binding motifs that have been identified in other CoA-SH/acyl-CoA binding proteins. Further comparisons of biotin-dependent carboxylases with other groups of enzymes in the protein data bank reveal that this family of biotin enzymes has strong similarities in specific domains to a number of ATP-utilizing enzymes and to the lipoyl-containing enzymes. These structural homologies are so extensive as to be highly suggestive of evolutionary relationships between biotin carboxylases and these other enzymes.     

Preparation and characterization of pancreatic lipase immobilized in Eudragit-matrix

Pancreatic lipase (EC 3.1.1.3) was immobilized by entrapping in a commercial preparation of acrylic/methacrylic acid ester-based copolymer (Eudragit E 30 D). The activity of the immobilized lipase beads with a diameter of 1.5-2.0 mm was found to be lower than that of the free lipase. The optimum pH was shifted to the alkaline region and the thermal stability increased, whereas the optimum temperature level remained unchanged. The most important reason for the decreased activity was diffusion limitations. The diffusion of the substrate and products became more pronounced, and lipolytic activity increased upon addition of n-hexane into the reaction medium. The storage and operational stabilities of the immobilized lipase were investigated, and both characteristics were found to be increased when compared to the free enzyme. Furthermore, mechanical or magnetic stirring during the operation were found to have no influence on the carrier-matrix as determined by nephelometric measurements.     

N-terminal and C-terminal domains of arrestin both contribute in binding to rhodopsin

Visual arrestin terminates the signal amplification cascade in photoreceptor cells by blocking the interaction of light activated phosphorylated rhodopsin with the G-protein transducin. Although crystal structures of arrestin and rhodopsin are available, it is still unknown how the complex of the two proteins is formed. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores (Alexa 633). The binding was recorded by time-resolved light scattering. To accomplish site-specific shielding of protein regions, in a first step all three wild-type cysteines were replaced by alanines. Nevertheless, regarding the magnitude and specificity of rhodopsin binding, the protein is still fully active. In a second step, new cysteines were introduced at selected sites to allow covalent binding of fluorophores. Upon attachment of Alexa 633 to the recombinant cysteines we observed that these bulky labels residing in the concave area of either the N- or the C-terminal domain do not perturb the activity of arrestin. By simultaneously modifying both domains with one Alexa 633 the binding capacity was reduced. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. This observation indicates that both concave sites participate in binding.     

The N-terminal domain of apolipoprotein B-100: structural characterization by homology modeling

Background  

Apolipoprotein B-100 (apo B-100) stands as one of the largest proteins in humans. Its large size of 4536 amino acids hampers the production of X-ray diffraction quality crystals and hinders in-solution NMR analysis, and thus necessitates a domain-based approach for the structural characterization of the multi-domain full-length apo B.     

Chemistry of tantalum clusters in solution and on SiO2 supports: analogies and contrasts

Tantalum clusters have been synthesized from Ta(CH2Ph)5 on the surface of porous fumed SiO2. When these clusters are small, incorporating, on average, several Ta atoms, their chemistry is similar to that of molecular tantalum clusters (and other early transition-metal) clusters. For example, The Ta-Ta bonds in these small supported clusters have been characterized by extended X-ray absorption fine structure (EXAFS), IR, and UV-vis spectroscopy, being similar to those in molecular analogues. The redox reactions of the supported clusters, characterized by X-ray absorption near-edge structure, are analogous to those of early transition-metal clusters in solution. In contrast to the largest of these clusters in solution and in the solid state, those supported on SiO2 are raftlike, facilitating the substantial metal-support-oxygen bonding that is evident in the EXAFS spectra. Samples consisting of tantalum clusters on SiO2 catalyze alkane disproportionation and the conversion of methane with n-butane to give other alkanes, but catalytic properties of analogous clusters in solution have barely been explored.     

Kinetic properties of dromedary pancreatic lipase: A comparative study on emulsified and monomolecular substrate

We demonstrated the use of X-ray photoelectron spectroscopy (XPS) to study DNA hybridization. Target DNA labeled with hexachloro-fluorescein (HEX) was hybridized to DNA arrays with four different probes. Each probe dot of the hybridized arrays was detected with XPS. The XPS Cl2p peak areas were found to decrease with an increase in mismatched bases in DNA probes. The Cl2p core-level peak area ratio of a probe perfectly matched to one, two and three base-mismatched probes accorded well with the results of conventional fluorescent imaging, which shows that XPS is a potential tool for analyzing DNA arrays. The DNA arrays’ hybridization efficiency was assessed by the molar ratio of chlorine to phosphorus in a DNA strand, which was determined from the relevant XPS Cl2p and P2p core-level peak areas after hybridization. This could provide a new method to detect DNA hybridization efficiency.     

Acyclic phenylalkanediols as substrates for the study of enzyme recognition. Regioselective acylation by porcine pancreatic lipase: a structural hypothesis for the enzymatic selectivity

The regioselective acylation of phenylalkanediols catalysed by porcine pancreatic lipase (PPL) was the reaction used for modelling different areas in the active site of the enzyme. With this aim, different racemic or prochiral (1,n)-diols, with n ranging from 2 to 6 were resolved via transesterification with vinyl acetate, and the results were explained according to microcrystalline enzyme structure. Thus, we describe a logical model for explaining the enzyme regio and stereoselectivity, based on three residues of the active site (Ser153, Phe216 and His264) which turned out to be crucial for the substrate binding and transformation.     

Synthesis and study of a lipophilic alpha-keto amide inhibitor of pancreatic lipase

[reaction: see text] A lipophilic alpha-keto amide, inhibitor of pancreatic lipase, was synthesized using a lipidic 2-amino alcohol as backbone. The chiral key intermediate 2-(tert-butyloxycarbonylamino)-D-undecen-5-ol was synthesized starting from D-glutamic acid. The inhibitor formed a stable monomolecular film at the air/water interface as shown by a force/area curve. Inhibition studies using the monomolecular film technique with mixed films of 1,2-dicaprin containing variable proportions of the inhibitor showed a 50% decrease in lipase activity at a 0.14 molar fraction.     

Medicinal plant phytochemicals and their inhibitory activities against pancreatic lipase: molecular docking combined with molecular dynamics simulation approach

Obesity is the worst health risk worldwide, which is linked to a number of diseases. Pancreatic lipase is considered as an affective cause of obesity and can be a major target for controlling the obesity. The present study was designed to find out best phytochemicals against pancreatic lipase through molecular docking combined with molecular dynamics (MD) simulation. For this purpose, a total of 3770 phytochemicals were docked against pancreatic lipase and ranked them on the basis of binding affinity. Finally, 10 molecules (Kushenol K, Rosmarinic acid, Reserpic acid, Munjistin, Leachianone G, Cephamycin C, Arctigenin, 3-O-acetylpadmatin, Geniposide and Obtusin) were selected that showed strong bonding with the pancreatic lipase. MD simulations were performed on top five compounds using AMBER16. The simulated complexes revealed stability and ligands remained inside the binding pocket. This study concluded that these finalised molecules can be used as drug candidate to control obesity.     

Noncovalent template-assisted mimicry of multiloop protein surfaces: assembling discontinuous and functional domains

We report here a novel noncovalent synthetic strategy for template-assembled de novo protein design. In this approach, a peptide was first conjugated with two oligoguanosine strands via click chemistry and the conjugates were then self-assembled in the presence of metal ions. G-quadruplex formation directs two peptide strands to assemble on one face of the scaffold and form an adjacent two loop surface. This approach can be used to rapidly prepare multiple two-loop structures with both homo- and heterosequences.     

Porcine pancreatic lipase mediated regio- and stereoselective hydrolysis: chemoenzymatic synthesis of (2S,3S)-2-amino-3,4-dihydroxybutyric acid

Porcine pancreatic lipase was used in the chemoenzymatic hydrolysis of 2-azido-3-hydroxy-4-methylcarbonyloxybutyl acetate. The reaction occurred with high regio- and stereoselectivity to give enantiomerically pure (2S,3R)-3-azido-2,4-dihydroxy butyl acetate 5 (e.e. >99%) which was easily converted to (2S,3S)-2-amino-3,4-dihydroxybutyric acid 1, an important synthetic intermediate in the synthesis of β-lactam antibiotics and phytosiderophores.     

Two-dimensional electronic conjugation: statics and dynamics at structural domains beyond molecular wires

Chemical architectures supporting a high degree of electronic conjugation serve as important functional components in devices and materials for advanced electronic and photonic applications. Increasing the spatial dimensionality of such constructs can fundamentally modify their optoelectronic properties and significantly alter intra- and intermolecular interactions that are crucial for understanding and controlling charge/energy-transfer processes. In this article, emerging design principles in the construction of well-defined conjugated platforms beyond molecular wires are highlighted. Both covalent and noncovalent approaches can be strategically employed to position one-dimensional (1D) substructures in a spatially well-defined manner in order to enhance both structural and functional complexity in a two-dimensional (2D) setting. A predictable and controllable switching mechanism can be designed and implemented with mobile 2D electronic conjugation that operates by correlated motions of inherently rigid 1D subunits. This emerging "dynamic" approach complements and challenges the prevailing "static" paradigm of conjugated chemical architectures.     

The functional domains of dopamine transporter for cocaine analog, CFT binding

Cocaine analogue, CFT (2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane) binding to dopamine transporter (DAT) in different species is quite heterogeneous. CFT is scarcely detected in bovine DAT whereas it is conspicuous in humans. To examine the structural basis for this functional discrepancy, we analyzed transporter chimeras of these two DATs. The CFT binding activities are avid in all of the chimeric DATs of which both of the 3rd and the 6-8th transmembrane domain (TM) are composed of human DAT sequences. On the contrary, CFT binding activities were scarcely detected if either or both of two regions are replaced with bovine sequences. These findings indicate that the CFT binding absolutely requires human DAT sequences, at least, in the regions encompassing the 3rd and 6-8th transmembrane domain (TM), and that these regions might contribute to form the 3-dimensional pocket for CFT binding.