Formation Of Higher Oligomers Research Articles

Non-enzymatic glycation of human angiogenin: Effects on enzymatic activity and binding to hRI and DNA

The effects of non-enzymatic glycation on the structural and functional properties of human angiogenin (hAng) have been investigated with respect to the formation of advanced glycated end products (AGEs), on prolonged treatment with d-Glucose, d-Fructose and d-Ribose at 37 °C. Fluorescence studies show the formation of fluorescent AGEs which exhibit emission maxima at 406 nm and 435 nm. Glycation of hAng with ribose leads to the maximum loss of its functional characteristic properties, as compared to fructose and glucose, along with the formation of higher oligomers. An increase in the incubation time results in the formation of higher oligomers with a concomitant decrease in the ribonucleolytic activity. The increase in the hydrodynamic radii of the glycated samples compared to native hAng is indicative of structural perturbations. The ribonucleolytic activity and the DNA binding ability of glycated hAng has been investigated by an agarose gel-based assay. Glycated hAng was unable to bind with human placental ribonuclease inhibitor (hRI), otherwise known to form one of the strongest protein-protein interaction systems with an affinity in the femtomolar range.

Selective Ethylene Dimerization by Palladium(II) Complexes Bearing a Phosphinoferrocene Sulfonate Ligand

Palladium(II) complexes featuring the hybrid anionic ligand 1′-(diphenylphosphino)ferrocene-1-sulfonate (L–), viz., trans-(Et3NH)2[Pd(μ-Cl)Me(L-κP)]2 (1), [Pd(Me)(dmap-κN1)(L-κ2O,P)](2; dmap = 4-(dimethylamino)pyridine), and [Pd(η3-allyl)(L-κ2O,P)] (6), were synthesized and together with the previously reported compounds trans-(Et3NH)2[PdCl2(L-κP)2] and [Pd(LCY)(L-κ2O,P)] (LCY = 2-[(dimethylamino-κN)methyl]phenyl-κC1 and 2-[(methylthio-κS)methyl]phenyl-κC1) tested as precatalysts for Pd-catalyzed ethylene dimerization. Only compound 1 gave rise to an active catalyst after activation by sequential halogen removal with Tl[PF6] and Na[BAr′4] (Ar′ = 3,5-bis(trifluoromethyl)phenyl) in chloroform. Thus, the formed catalyst efficiently mediated the dimerization of ethylene showing both good activity (TOF ≈ 95 h–1) and high selectivity for 1-butene (95%) at 21 °C and 30 bar of ethylene pressure. DFT calculations have shown that the dimerization reaction is thermodynamically preferred over the formation of higher oligomers and that O,P-chelate coordination of the phosphinosulfonate ligand in all Pd(II) reaction intermediates is vital for the catalytic process. In particular, the O,P-chelating phosphinoferrocene sulfonate ligand stabilizes and electronically differentiates the reaction intermediates and favors concerted ethyl migration to coordinated ethylene giving rise to 1-butene.

The crystal structure of an essential high-temperature requirement protein HtrA1 (Rv1223) from Mycobacterium tuberculosis reveals its unique features.

High-temperature requirement A (HtrA) proteins, which are members of the heat-shock-induced serine protease family, are involved in extracytoplasmic protein quality control and bacterial survival strategies under stress conditions, and are associated with the virulence of several pathogens; they are therefore major drug targets. Mycobacterium tuberculosis possesses three putative HtrAs: HtrA1 (Rv1223), HtrA2 (Rv0983) and HtrA3 (Rv0125). Each has a cytoplasmic region, a transmembrane helix and a periplasmic region. Here, the crystal structure of the periplasmic region consisting of a protease domain (PD) and a PDZ domain from an M. tuberculosis HtrA1 mutant (mHtrA1S387A) is reported at 2.7 Å resolution. Although the mHtrA1S387A PD shows structural features similar to those of other HtrAs, its loops, particularly L3 and LA, display different conformations. Loop L3 communicates between the PDs of the trimer and the PDZ domains and undergoes a transition from an active to an inactive conformation, as reported for an equivalent HtrA (DegS). Loop LA, which is responsible for higher oligomer formation owing to its length (50 amino acids) in DegP, is very short in mHtrA1S387A (five amino acids), as in mHtrA2 (also five amino acids), and therefore lacks essential interactions for the formation of higher oligomers. Notably, a well ordered loop known as the insertion clamp in the PDZ domain interacts with the protease domain of the adjacent molecule, which possibly aids in the stabilization of a trimeric functional unit of this enzyme. The three-dimensional structure of mHtrA1S387A presented here will be useful in the design of enzyme-specific antituberculosis inhibitors.

Light-Induced RAFT Single Unit Monomer Insertion in Aqueous Solution-Toward Sequence-Controlled Polymers.

First report on the sequential, visible light-initiated, single unit monomer insertion (SUMI) of N,N-dimethylacrylamide (DMAm) into the reversible addition fragmentation chain transfer (RAFT) agent, 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CTA1 ), in aqueous solution is provided. The specificity for SUMI over formation of higher oligomers and/or RAFT agent-derived by-products is higher for longer irradiation wavelengths. Red light provides the cleanest product (selective SUMI), showing a linear pseudo-first order kinetic profile to high (>80%) conversion, but also the slowest reaction rate. Blue light provides a relatively rapid reaction, but also gives some by-products (<2%) and the kinetic profile displays a conversion plateau at >65% conversion. Higher specificity with red light is attributed to CTA1 absorbing at longer wavelengths than the SUMI product, which allows selective excitation of CTA1 . The use of a higher reaction temperature (65 °C vs ambient) results in a higher reaction rate and a reduction in oligomer formation.

A unique cysteine-rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, used in DNA synthesis and repair. Two different mechanisms help deliver the required electrons to the RNR active site. Formate can be used as reductant directly in the active site, or glutaredoxins or thioredoxins reduce a C-terminal cysteine pair, which then delivers the electrons to the active site. Here, we characterized a novel cysteine-rich C-terminal domain (CRD), which is present in most class II RNRs found in microbes. The NrdJd-type RNR from the bacterium Stackebrandtia nassauensis was used as a model enzyme. We show that the CRD is involved in both higher oligomeric state formation and electron transfer to the active site. The CRD-dependent formation of high oligomers, such as tetramers and hexamers, was induced by addition of dATP or dGTP, but not of dTTP or dCTP. The electron transfer was mediated by an array of six cysteine residues at the very C-terminal end, which also coordinated a zinc atom. The electron transfer can also occur between subunits, depending on the enzyme's oligomeric state. An investigation of the native reductant of the system revealed no interaction with glutaredoxins or thioredoxins, indicating that this class II RNR uses a different electron source. Our results indicate that the CRD has a crucial role in catalytic turnover and a potentially new terminal reduction mechanism and suggest that the CRD is important for the activities of many class II RNRs.

Synthesis of Two 2,2′-Bipyridine Containing Macrocycles for the Preparation of Interlocked Architectures

The synthesis and characterisation of two 28-membered, 2,2′-bipyridine-containing macrocycles in high yield is reported. The first imine-containing macrocycle was formed via a Williamson ether synthesis and showed no evidence of higher oligomer formation. Reduction of the imines with sodium borohydride produced the second macrocycle quantitatively.

Progress in Studies on the RNA World

The montmorillonite-catalyzed reactions of D, L-ImpA with D, L-ImpU generates RNA-like oligomers. The structures of the dimers to pentamers were investigated and homochiral products were identified in greater amounts than would be expected if theoretical amounts of each were formed. The homochirality increased from 64% to 97% as the chain length increased from dimers to pentamers. Investigation of the effect of pH, occupancy of the interlayer space and the influence of various cations in the reaction provided further insight into physical process in the mechanism of the catalysis. A detailed analysis of dimers was carried out in view of there being key intermediates towards formation of higher oligomers. The study was extended to the synthesis of non-standard dimers including those formed with deoxy-ribonucleotides.

Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS

Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A-H2B dimer or H3-H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein-protein interactions in solution.

Insights into Protein Sequence and Structure-Derived Features Mediating 3D Domain Swapping Mechanism using Support Vector Machine Based Approach

3-dimensional domain swapping is a mechanism where two or more protein molecules form higher order oligomers by exchanging identical or similar subunits. Recently, this phenomenon has received much attention in the context of prions and neurodegenerative diseases, due to its role in the functional regulation, formation of higher oligomers, protein misfolding, aggregation etc. While 3-dimensional domain swap mechanism can be detected from three-dimensional structures, it remains a formidable challenge to derive common sequence or structural patterns from proteins involved in swapping. We have developed a SVM-based classifier to predict domain swapping events using a set of features derived from sequence and structural data. The SVM classifier was trained on features derived from 150 proteins reported to be involved in 3D domain swapping and 150 proteins not known to be involved in swapped conformation or related to proteins involved in swapping phenomenon. The testing was performed using 63 proteins from the positive dataset and 63 proteins from the negative dataset. We obtained 76.33% accuracy from training and 73.81% accuracy from testing. Due to high diversity in the sequence, structure and functions of proteins involved in domain swapping, availability of such an algorithm to predict swapping events from sequence and structure-derived features will be an initial step towards identification of more putative proteins that may be involved in swapping or proteins involved in deposition disease. Further, the top features emerging in our feature selection method may be analysed further to understand their roles in the mechanism of domain swapping.

Solid-Phase Random Glycosylation

Two different approaches were employed to study solid phase random glycosylations to obtain oligosaccharide libraries. In approach I, Wang resin esters were attached to the acceptors structures. Following their glycosylation and resin cleavage, the peracetylated components of the oligosaccharide libraries were characterized. In approach II, polymer-linked donor components could be employed and processed correspondingly. Approach I proved to be superior regarding yield and versatility of products and also allowed the formation of higher oligomers.

Structural Properties of AMP-activated Protein Kinase: DIMERIZATION, MOLECULAR SHAPE, AND CHANGES UPON LIGAND BINDING

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by approximately 5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr(172), thus positively affecting AMPK activity.

The Crystal Structure of β-Alanine Synthase from Drosophila melanogaster Reveals a Homooctameric Helical Turn-Like Assembly

β-Alanine synthase (βAS) is the third enzyme in the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of the nucleotide bases uracil and thymine in higher organisms. It catalyzes the hydrolysis of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyrate to the corresponding β-amino acids. βASs are grouped into two phylogenetically unrelated subfamilies, a general eukaryote one and a fungal one. To reveal the molecular architecture and understand the catalytic mechanism of the general eukaryote βAS subfamily, we determined the crystal structure of Drosophila melanogaster βAS to 2.8 Å resolution. It shows a homooctameric assembly of the enzyme in the shape of a left-handed helical turn, in which tightly packed dimeric units are related by 2-fold symmetry. Such an assembly would allow formation of higher oligomers by attachment of additional dimers on both ends. The subunit has a nitrilase-like fold and consists of a central β-sandwich with a layer of α-helices packed against both sides. However, the core fold of the nitrilase superfamily enzymes is extended in D. melanogaster βAS by addition of several secondary structure elements at the N-terminus. The active site can be accessed from the solvent by a narrow channel and contains the triad of catalytic residues (Cys, Glu, and Lys) conserved in nitrilase-like enzymes.

Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

Transcriptional initiation invariably involves the transition from a closed RNA polymerase (RNAP) promoter complex to a transcriptional competent open complex. Activators of the bacterial sigma(54)-RNAP are AAA+ proteins that couple ATP hydrolysis to restructure the sigma(54)-RNAP promoter complex. Structures of the sigma(54) activator PspF AAA+ domain (PspF(1-275)) bound to sigma(54) show two loop structures proximal to sigma(54) as follows: the sigma(54) contacting the GAFTGA loop 1 structure and loop 2 that classifies sigma(54) activators as pre-sensor 1 beta-hairpin AAA+ proteins. We report activities for PspF(1-275) mutated in the AAA+ conserved sensor I threonine/asparagine motif (PspF(1-275)(T148A), PspF(1-275)(N149A), and PspF(1-275)(N149S)) within the second region of homology. We show that sensor I asparagine plays a direct role in ATP hydrolysis. However, low hydrolysis rates are sufficient for functional output in vitro. In contrast, PspF(1-275)(T148A) has severe defects at the distinct step of sigma(54) promoter restructuring. This defect is not because of the failure of PspF(1-275)(T148A) to stably engage with the closed sigma(54) promoter, indicating (i) an important role in ATP hydrolysis-associated motions during energy coupling for remodeling and (ii) distinguishing PspF(1-275)(T148A) from PspF(1-275) variants involved in signaling to the GAFTGA loop 1, which fail to stably engage with the promoter. Activities of loop 2 PspF(1-275) variants are similar to those of PspF(1-275)(T148A) suggesting a functional signaling link between Thr(148) and loop 2. In PspF(1-275) this link relies on the conserved nucleotide state-dependent interaction between the Walker B residue Glu(108) and Thr(148). We propose that hydrolysis is relayed via Thr(148) to loop 2 creating motions that provide mechanical force to the GAFTGA loop 1 that contacts sigma(54).

Evidence for a dimeric assembly of two titin/telethonin complexes induced by the telethonin C-terminus

The Z-disk region defines the lateral boundary of the sarcomere and requires a high level of mechanical strength to provide a stable framework for large filamentous muscle proteins. The level of complexity at this area is reflected by a large number of protein–protein interactions. Recently, we unraveled how the N-terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the N-terminal titin-binding segment of the Z-disk ligand telethonin/T-cap [Zou, P., Pinotsis, N., Lange, S., Song, Y.H., Popov, A., Mavridis, I., Mayans, O.M., Gautel, M., Wilmanns, M., 2006. Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. Nature 439, 229–233]. In this contribution, we present structural data of a related complex of the titin N-terminus with full-length telethonin. The C-terminus of telethonin remains invisible, suggesting that it does not fold into a defined structure even in the presence of titin. In contrast to the structure with truncated telethonin, a dimer of two titin/telethonin complexes is formed within the crystal environment, potentially indicating the formation of higher oligomers. We further investigated the structure and dynamics of this assembly by small-angle X-ray scattering, circular dichroism, and in vivo complementation data. The data consistently indicate the involvement of the C-terminal part of telethonin into the assembly of two titin/telethonin complexes.

Nucleotide binding and filament assembly of recombinant yeast septin complexes

Septins are filament-forming GTPases involved in cytokinesis and cortical organization. In the yeast Saccharomyces cerevisiae, the septins encoded by CDC3, CDC10, CDC11, and CDC12 form a high-molecular-weight complex, localized at the cytoplasmic face of the plasma membrane in the mother-bud neck. While septin function at the cellular level is fairly well understood, progress on structure-function analysis of these proteins has been slow and limited by the lack of large amounts of pure complex. While monomeric septins form apparently non-native aggregates, stable recombinant complexes of two, three, or four yeast septins can be produced by co-expression from bi-cistronic vectors in E. coli. The septin polypeptides show various degrees of saturation with guanine nucleotides in different complexes. The binary core Cdc3p-Cdc12p complex contains no bound nucleotide. While ternary complexes are partially saturated and can bind extraneously added nucleotide with micromolar affinity, only the complete four-component septin complex is fully coordinated with tightly bound GDP/GTP after chromatographic purification. We show here that the nucleotide-binding sites of the septins show drastic changes on formation of higher oligomers. Although the binary core Cdc3p-Cdc12p complex does not form filaments, the ternary and quaternary complexes form bundles of paired filaments. In the case of ternary complexes, filament formation is stimulated by guanine nucleotide, but is not dependent on the presence or absence of the gamma-phosphate.

ATP-dependent Transcriptional Activation by Bacterial PspF AAA+Protein

Transcription activation by bacterial σ 54-dependent enhancer-binding proteins (EBPs) requires their tri-nucleotide hydrolysis to restructure the σ 54 RNA polymerase (RNAP). EBPs share sequence similarity with guanine nucleotide binding-proteins and ATPases associated with various cellular activities (AAA) proteins, especially in the mononucleotide binding P-loop fold. Using the phage shock protein F (PspF) EBP, we identify P-loop residues responsible for nucleotide binding and hydrolysis, consistent with their roles in other P-loop NTPases. We show the refined low-resolution structure of an EBP, PspF, revealing a hexameric ring organisation characteristic of AAA proteins. Functioning of EBPs involves ATP binding, higher oligomer formation and ATP hydrolysis coupled to the restructuring of the RNAP. This is thought to be a highly coordinated multi-step process, but the nucleotide-driven mechanism of oligomerisation and ATP hydrolysis is little understood. Our kinetic and structural data strongly suggest that three PspF dimers assemble to form a hexamer upon nucleotide binding. During the ATP hydrolysis cycle, both ATP and ADP are bound to oligomeric PspF, in line with a sequential hydrolysis cycle. We identify a putative R-finger, and show its involvement in ATP hydrolysis. Substitution of this arginine residue results in nucleotide-independent formation of hexameric rings, structurally linking the putative R-finger and, by inference, a specific nucleotide interaction to the control of PspF oligomerisation.

Synthesis and X-ray crystal structure of polymeric and dimeric copper(I) cyanide complexes incorporating a bicyclic guanidine ligand

1,3,4,6,7,8-Hexahydro-2 H-pyrimido[1,2- a]pyrimidine (hppH) reacts with copper(I) cyanide to afford the polymeric chain [(hppH)CuCN] ∞ ( 1), consisting of distorted trigonal planar copper atoms linked by cyanide bridges. Attempts to isolate molecular species by repeating the reaction in the presence of triphenylphosphine gave a dimeric complex of the general formula [Cu 2(CN) 2(PPh 3) 4(hppH)] ( 2). The solid-state structure of 2 is consistent with a zwitterionic complex, comprising of [Cu (+)(PPh 3) 2(hppH)] and [Cu (−)(CN) 2(PPh 3) 2] metal centres, linked by a cyanide bridge. Variable temperature NMR studies suggest that the formation of higher oligomers may occur in solution at low temperature.

Dimerization of human growth hormone in the presence of metal ions.

Zn(2+) and Co(2+) ions are known to promote human growth hormone reversible dimerization. In these studies, dimerization was also shown to be initiated by nine other metal ions: Cd(2+), Hg(2+), Cu(2+), Ag+, Au(3+), Au+, Pd(2+), Ni(2+), and Pt(4+). In some cases (Hg(2+), Ag(+), Au(3+), and Ni(2+)) formation of higher oligomers also took place. In addition further detailed investigation of dimerization in the presence of Zn(2+) ions was carried out.

Low temperature measurements of the rate of association to benzene dimers in helium

The association of benzene molecules to form dimers has been studied at temperatures between 15 and 123 K in a CRESU (Cinétique de Réactions en Ecoulement Supersonique Uniforme) apparatus with helium as the buffer gas. Second-order rate coefficients (k2) for the formation of dimers have been determined for temperatures between 15 and 88 K. The effective third-order rate coefficients (no study of fall off behavior has been undertaken) obtained by dividing the values of k2 by [He] show a strong negative temperature dependence. Further evidence for the formation of dimers at critical concentrations of benzene has been obtained from a study of the CH+C6H6 reaction and from LIF spectra. Modelling calculations indicate that any systematic errors in the results arising from the formation of higher oligomers are small. Based on a model in which it is assumed that the benzene molecules in the dimer are free to rotate, third-order rate coefficients have been calculated for dimerization and are in good agreement with the experimental values for T⩾30 K, where effects of falloff should not be too great.

Alpha beta protomers of Na+,K+-ATPase from microsomes of duck salt gland are mostly monomeric: Formation of higher oligomers does not modify molecular activity

The distance that separates alphabeta protomers of the Na(+), K(+)-ATPase in microsomes and in purified membranes prepared from duck nasal salt glands was estimated by measuring fluorescence resonance energy transfer between anthroylouabain bound to a population of alphabeta protomers and either N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain or 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain bound to the rest. Energy transfer between probes bound in the microsomal preparation was less than in the purified membranes. The efficiency of energy transfer between anthroylouabain and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain was 29.2% in the microsomes compared with 62.6% in the purified preparation. Similar results were obtained with 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain as acceptor. We calculate that either the protomer bound probes were on the average 13 A farther apart in the microsomes than in the purified membranes, or that 53% of the protomers are monomeric in the microsome preparation. Microsomes prepared in the presence of phalloidin (a toxin that binds to F actin and stabilizes the actin-based cytoskeleton) showed less quench than those prepared in its absence. The data support the hypothesis that protomers are kept apart by their association with the cytoskeleton. The turnover rate while hydrolyzing ATP is the same in the microsomal and purified preparations; higher oligomer formation has no significant effect on the enzyme reaction mechanism.