Intramolecular signaling in immunoglobulins -- new evidence emerging from the use of supramolecular protein ligands.

The postulated intramolecular signaling in immunoglobulins generated by antigen binding has been controversial for years. The high heterogeneity of immune complexes as signaling systems and the requirement of the immobilized antigen form for efficient triggering of effector activity is likely the reason for the lack of clarity. Here we present new evidence supporting the notion of intramolecular signaling, based on the use of supramolecular dyes that bind to signal-derived specific sites in immunoglobulins.     

l-Arginine induces protein aggregation and transformation of supramolecular structures of the aggregates

Protein misfolding, self-assembly, and aggregation are an essential problem in cell biology, biotechnology, and biomedicine. The protein aggregates are very different morphologically varying from soluble amorphous aggregates to highly ordered amyloid-like fibrils. The objective of this study was to elucidate the role of the amino acid l-arginine (Arg), a widely used suppressor of protein aggregation, in the regulation of transformations of soluble aggregation-prone proteins into supramolecular structures of higher order. However, a striking potential of Arg to govern the initial events in the process of protein aggregation has been revealed under environment conditions where the protein aggregation in its absence was not observed. Using dynamic light scattering we have demonstrated that Arg (10–100 mM) dramatically accelerated the dithiothreitol-induced aggregation of acidic model proteins. The inhibitory effect on the protein aggregation was revealed at higher concentrations of Arg. Using atomic force microscopy it was shown that aggregation of α-lactalbumin from bovine milk induced upon addition of Arg reached a state of formation of supramolecular structures of non-fibrillar species profoundly differing from those of the individual protein in type, size, and shape. The interaction of another positively charged amino acid l-lysine with α-lactalbumin also resulted in profound acceleration of the aggregation process and transformation of supramolecular structures of the aggregates.     

1,4-Dihydropyridines as calcium channel ligands and privileged structures

1. The 1,4-dihydropyridine nucleus serves as the scaffold for important cardiovascular drugs—calcium antagonists—including nifedipine, nitrendipine, amlodipine, and nisoldipine, which exert their antihypertensive and antianginal actions through actions at voltage-gated calcium channels of the Ca_V1 (L-type) class.2. These drugs act at a specific receptor site for which defined structure-activity relationships exist, including stereoselectivity.3. Despite the widespread occurrence of the Ca_V1 class of channel, the calcium antagonists exhibit significant selectivity of action in the cardiovascular system. This selectivity arises from a number of factors including subtype of channel, state-dependent interactions, pharmacokinetics, and mode of calcium mobilization.4. The 1,4-dihydropyridine nucleus is also a privileged structure or scaffold that can, when appropriately decorated substituents, interact at diverse receptors and ion channels, including potassium and sodium channels and receptors of the G-protein class.     

The use and development of the dynamic light-scattering method to investigate supramolecular structures in aqueous solutions of bacterial lipopolysaccharides

The temperature dependence of the scattering intensity, average size, and size distribution for supramolecular particles in aqueous solutions of lipopolysaccharides from Azospirillum bacteria was investigated by dynamic light scattering. Relationships were obtained that made it possible to comparatively estimate the mass–volume concentration of the biopolymeric substance in suspensions and the number concentration of supramolecular particles with their size and degree of polydispersity taken into account. In the range from 0 to 60°C, two types of the temperature dependence of scattering intensity were found: (a) with an irregular spasmodic change in scattering intensity and with considerable heterogeneity of the systems with respect to particle size and (b) with a smoother character of this dependence with considerably decreased heterogeneity of the suspensions. In the ranges of the latter type, whose location depended on what strain was used to isolate lipopolysaccharides, it proved to be possible to correctly determine the parameters of the supramolecular particles (of the supposedly formed micellar phase) by dynamic light scattering. The revealed statistically significant differences in the size and the concentration of the micellar particles are explained by their dependence on the peculiarities of the chemical structure of lipopolysaccharides. Atomic-force microscopy was used for an independent morphological estimation of the preparations, yielding good agreement with the dynamic light-scattering results.     

Designing supramolecular protein assemblies

Many natural proteins self-assemble, either to fulfill their biological function or as part of a pathogenic process. Biological assembly phenomena such as amyloidogenesis, domain swapping and symmetric oligomerization are inspiring new strategies for designing proteins that self-assemble to form supramolecular complexes. Recent advances include the design of novel proteins that assemble into filaments, symmetric cages and regular arrays.     

Double-stranded nucleic acids in liquid-crystalline dispersions as building blocks for cross-linked supramolecular structures

Double-stranded DNA fixed in a cholesteric liquid-crystalline dispersion was used for generating an ordered supramolecular structure in the presence of anthracycline and copper (II) ions. The structure is stable in a water-salt solution and does not require poly(ethyleneglycol).The ordered network can be immobilized on the surface of a polymeric film, and may collapse in the presence of biologically and pharmacologically relevant compounds. Accordingly, the DNA-based liquid-crystalline network represents the basis to obtain novel highly sensitive biosensing units.     

The use of ubiquitin-peptide extensions as protein kinase substrates

Four ubiquitin-peptide extensions prepared as cloned products in E. coli were tested as casein kinase II substrates. Two extensions containing the sequence Ser-Glu-Glu-Glu-Glu-Glu were readily phosphorylated by partially purified rabbit reticulocyte casein kinase II. The other two fusion proteins, which lack a consensus phosphorylation site for casein kinase II, did not serve as substrates under identical reaction conditions. Native ubiquitin was not phosphorylated by reticulocyte casein kinase II, nor have we observed its phosphorylation in crude extracts from HeLa cells, mouse liver, or Xenopus eggs. Ubiquitin's apparent lack of phosphorylatable residues coupled with its remarkable heat stability and rapid migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels make the protein an attractive carrier for carboxyl-terminal peptides containing specific phosphorylation sites. Such ubiquitin extension proteins should prove valuable as protein kinase substrates.     

Junctions between i-motif tetramers in supramolecular structures

The symmetry of i-motif tetramers gives to cytidine-rich oligonucleotides the capacity to associate into supramolecular structures (sms). In order to determine how the tetramers are linked together in such structures, we have measured by gel filtration chromatography and NMR the formation and dissociation kinetics of sms built by oligonucleotides containing two short C stretches separated by a non-cytidine-base. We show that a stretch of only two cytidines either at the 3'- or 5'-end is long enough to link the tetramers into sms. The analysis of the properties of sms formed by oligonucleotides differing by the length of the oligo-C stretches, the sequence orientation and the nature of the non-C base provides a model of the junction connecting the tetramers in sms.     

Stress protein of cyanobacteria CP36: interaction with photoactive complexes and formation of supramolecular structures

Cyanobacterium Anacystis nidulans R2, Synechocystis sp. PCC 6803 (wild-type strain and mutants Delta2 and Delta3 lacking PSII and PSI, respectively), and Synechocystis sp. BO 9201 synthesize the pigment--protein complex CP36 (CPIV-4, CP43') under iron deficiency in the medium. Accumulation of CP36 is accompanied by structural reorganizations in the photosynthetic membranes. Integrating mean times of excitation relaxation (quenching) are 2.2 nsec (CP36), 1 nsec (PSI), and 420 psec (PSII in Fm state). The energy migration between CP36 and the photosystems can be described by a model of a one-layer ring of CP36 around core-complexes. The excitation from CP36 to PSI is transferred within <10 psec. The energy transfer from CP36 to PSII occurs during 170 psec. Cells with low content of CP36 probably contain only a latent fraction of unbound to phycobilisomes PSII which is the analog of PSIIbeta of higher plants. In PSI there are four binding sites for CP36 monomers per RC. PSII can bind up to 32 molecules of CP36 per RC. Cells with a large amount of CP36 contain monomer form of PSII core-complex which can bind eight tetramers of CP36 (8 binding sites). In conditions of iron deficiency only one monomer of a dimer PSII core-complex is destroyed and released chlorophyll is accumulated in CP36. Accumulation of CP36 in A. nidulans cells can be accompanied by membrane stacking which is similar to the stacking in chlorophyll b-containing organisms. The stacking can occur in the region of localization of PSII latent fraction bound to CP36. The membrane stacking shields PSII stromal surfaces from the aqueous phase for activation of electron transfer on the acceptor side of PSII.     

Bioadhesion of supramolecular structures at supported planar bilayers as studied by the quartz crystal microbalance

A quartz crystal microbalance (QCM) was used to study the adhesion behavior of supramolecular aggregates at supported planar bilayers (SPBs). The QCM technique is a suitable method to detect the adsorption of biomolecules at the quartz surface owing to its sensitivity for changes in mass and viscoelastic properties. To simulate biomembranes, the quartz plates were coated with highly ordered lipid films. Therefore, a combination of self-assembled monolayers and Langmuir-Blodgett films was used. Firstly, the adsorption of liposomes coupled with the lectin concanavalin A was investigated at glycolipid-containing model membranes. Using different carbohydrates, it was possible to determine specific and nonspecific parts of the interactions. The adhesion occurred owing to specific lectin-carbohydrate interactions (about 20%) and to nonspecific interactions (about 80%). The composition of the liposomes was changed to simulate the structure of a native biomembrane consisting of the glycocalix, the lipid-protein bilayer, and the cytoskeleton. An artificial glycocalix was created by incorporating poly(ethylene glycol) into the liposomes. Liposomes which were intravesicular polymerized with polyacrylamide or polyacrylcholate simulated the cytoskeleton. It was determined that the modified liposomes had significant lower interactions with SPBs. The adsorption was reduced by approximately 80% compared to unmodified liposomes. Secondly, a model was developed for the detection of interactions between simple or mixed bile salt micelles and model membranes. It was found that simple bile salts did not adsorb at model membranes. Binary systems consisting of bile salt and phospholipid induced only small interactions. On the other hand, ternary systems consisting of bile salt, phospholipid, and fatty acid showed strong interactions. A dependence on the chain length of the fatty acid was observed. Thirdly, the interaction between ganglioside-containing model membranes and cholera toxin (beta-subunit) was investigated. Different ganglioside fractions showed varying adsorption in the following sequence: GM1 > GD1a > GD1b > GT1b.     

Conformational dynamics of supramolecular protein assemblies

Supramolecular protein assemblies including molecular motors, cytoskeletal filaments, chaperones, and ribosomes play a central role in a broad array of cellular functions ranging from cell division and motility to RNA and protein synthesis and folding. Single-particle reconstructions of such assemblies have been growing rapidly in recent years, providing increasingly high resolution structural information under native conditions. While the static structure of these assemblies provides essential insight into their mechanism of biological function, their dynamical motions provide additional important information that cannot be inferred from structure alone. Here we present an unsupervised computational framework for the analysis of high molecular weight protein assemblies and use it to analyze the conformational dynamics of structures deposited in the Electron Microscopy Data Bank. Protein assemblies are modeled using a recently introduced coarse-grained modeling framework based on the finite element method, which is used to compute equilibrium thermal fluctuations, elastic strain energy distributions associated with specific conformational transitions, and dynamical correlations in distant molecular domains. Results are presented in detail for the ribosome-bound termination factor RF2 from Escherichia coli, the nuclear pore complex from Dictyostelium discoideum, and the chaperonin GroEL from E. coli. Elastic strain energy distributions reveal hinge-regions associated with specific conformational change pathways, and correlations in collective molecular motions reveal dynamical coupling between distant molecular domains that suggest new, as well as confirm existing, allosteric mechanisms. Results are publically available for use in further investigation and interpretation of biological function including cooperative transitions, allosteric communication, and molecular mechanics, as well as in further classification and refinement of electron microscopy based structures.     

The use of soluble protein structures in modeling helical proteins in a layered membrane

Major advances have been made in the prediction of soluble protein structures, led by the knowledge-based modeling methods that extract useful structural trends from known protein structures and incorporate them into scoring functions. The same cannot be reported for the class of transmembrane proteins, primarily due to the lack of high-resolution structural data for transmembrane proteins, which render many of the knowledge-based method unreliable or invalid. We have developed a method that harnesses the vast structural knowledge available in soluble protein data for use in the modeling of transmembrane proteins. At the core of the method, a set of transmembrane protein decoy sets that allow us to filter and train features recognized from soluble proteins for transmembrane protein modeling into a set of scoring functions. We have demonstrated that structures of soluble proteins can provide significant insight into transmembrane protein structures. A complementary novel two-stage modeling/selection process that mimics the two-stage helical membrane protein folding was developed. Combined with the scoring function, the method was successfully applied to model 5 transmembrane proteins. The root mean square deviations of the predicted models ranged from 5.0 to 8.8?Å to the native structures.     

Chemistry of molecular and supramolecular structures of vanadium(IV) and dioxygen-bridged V(V) complexes incorporating tridentate hydrazone ligands

Four hydrazone ligands: 2-benzoylpyridine benzoyl hydrazone (HBPB), di-2-pyridyl ketone nicotinoyl hydrazone (HDKN), quinoline-2-carbaldehyde benzoyl hydrazone (HQCB), and quinoline-2-carbaldehyde nicotinoyl hydrazone (HQCN) and four of their complexes with vanadyl salts have been synthesized and characterized. Single crystals of HBPB and complexes [VO(BPB)(μ₂-O)]₂ (1) and [VO(DKN)(μ₂-O)]₂·½H₂O (2) were isolated and characterized by X-ray crystallography. Each of the complexes exhibits a binuclear structure where two vanadium(V) atoms are bridged by two oxygen atoms to form distorted octahedral structures within cis-N₂O₄ donor sets. In most complexes, the uninegative anions function as tridentate ligands, coordinating through the pyridyl- and azomethine-nitrogen atoms and enolic oxygen whereas in complex [VO(HQCN)(SO₄)]SO₄·4H₂O (4) the ligand is coordinated in the keto form. Complexes [VO(QCB)(OMe)]·1.5H₂O (3) and 4 are found to be EPR active and showed well-resolved axial anisotropy with two sets of eight line pattern.     

The small protein CP12: a protein linker for supramolecular complex assembly

CP12 is an 8.5-kDa nuclear-encoded chloroplast protein, isolated from higher plants. It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12. The role of CP12 in this complex assembly has not been determined. To address this question, we cloned a cDNA encoding the mature CP12 from the green alga Chlamydomonas reinhardtii and expressed it in Escherichia coli. Sequence alignments show that it is very similar to other CP12s, with four conserved cysteine residues forming two disulfide bridges in the oxidized CP12. On the basis of reconstitution assays and surface plasmon resonance binding studies, we show that oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex, and we propose a model in which CP12 associates with GAPDH, causing its conformation to change. This GAPDH/CP12 complex binds PRK to form a half-complex (one unit). This unit probably dimerizes due partially to interactions between the enzymes of each unit. Reduced CP12 being unable to reconstitute the complex, we studied the structures of oxidized and reduced CP12 by NMR and circular dichroism to determine whether reduction induced structural transitions. Oxidized CP12 is mainly composed of alpha helix and coil segments, and is extremely flexible, while reduced CP12 is mainly unstructured. Remarkably, CP12 has similar physicochemical properties to those of "intrinsically unstructured proteins" that are also involved in regulating macromolecular complexes, or in their assembly. CP12s are thus one of the few protein families of intrinsically unstructured proteins specific to plants.     

The use of fluorescamine as a detection reagent in protein microcharacterization

With recent advances in protein microchemistry, compatible methods for the preparation and quantitation of proteins and peptides are required. Fluorescamine, a reagent which reacts with primary amino groups has been used successfully to detect amino acids, peptides, and proteins in various micromethods. This article discusses these methods which include (1) amino acid analysis of protein and peptide hydrolysates with postcolumn fluorescamine derivatization; (2) purification and characterization of proteins and peptides by reversed-phase HPLC with postcolumn fluorescamine derivatization; (3) purification of peptides by two-dimensional chromatography and electrophoresis on thin-layer cellulose with fluorescamine staining; and (4) electroblotting of protein bands from SDS-PAGE to glass fiber filters and polyvinylidene difluoride (PVDF) membranes with fluorescamine staining. In addition, this article also compares a postcolumn fluorescamine detection system with a UV detection system in the applications of amino acid analysis and reversed-phase HPLC protein/peptide analysis.     

Retinoids as ligands and coactivators of protein kinase C alpha.

Whereas retinoic acids control nuclear events, a second class of retinol metabolites, that is, the hydroxylated forms exemplified by 14-hydroxy-retro-retinol (HRR), operate primarily in the cytoplasm. They function as regulatory cofactors for cell survival/cell death decisions. In accordance with these biological aspects, we demonstrate that these retinoids bound protein kinase C (PKC) alpha with nanomolar affinity and markedly enhance the activation of PKC alpha and the entire downstream MAP kinase pathway by reactive oxygen species. HRR was 10 times more efficient than retinol, and the optimum doses are 10-7 and 10-6 M, respectively. PKC alpha activation was reversed rapidly by imposition of reducing conditions. The retinoid binding site was mapped to the first cysteine-rich region in the regulatory domain, C1A, yet was distinct from the binding sites of diacylglycerol and phorbol esters. The C1B domain bound retinoids poorly. The emerging theme is that retinoids serve as redox regulators of protein kinase C.     

Three Mn(II) supramolecular coordination complexes containing carboxylate-tetrazolate ligands

Three novel complexes [Mn(atza)₂(H₂O)₄] (1), [Mn(nptza)₂(CH₃OH)₄] (2), and [Mn(a4-ptz)₂(H₂O)₂]_n · 2nH₂O] (3) [atza = 5-aminotetrazole-1-acetato, nptza = 5-[(4-nitryl)phenyl] tetrazole-1-acetato, a4-ptz = 5-[N-acetato(4-pyridyl)] tetrazole] containing carboxylate-tetrazolate ligands have been synthesized and characterized by element analysis. X-ray crystallography shows that complexes 1 and 2 both contain mononuclear structure. The complex 3 is a 1D polymeric chain structure. Compounds 1-3 are self-assembled to form supramolecular structures through hydrogen bonds interactions.     

The hydration of protein secondary structures

The hydration of the main-chain carbonyl (CO) groups in proteins have been studied using infra-red spectroscopy, and computer-graphics analysis of high resolution protein crystal structures. The IR measurements indicate that the strength of water binding to the CO groups is lower in beta-sheet proteins compared with alpha-helical ones. Analysis of the protein crystal structures shows that this is due primarily to differences in the geometry of water-CO group interactions in the two types of secondary structure.     

Temperature affects the supramolecular structures resulting from alpha-lactalbumin-lysozyme interaction

The interaction between alpha-lactalbumin and lysozyme, two globular proteins with highly homologous tertiary structures but opposite electric charges, was investigated. As assessed by isothermal titration calorimetry, lysozyme did not bind to the native form of alpha-lactalbumin but did interact with calcium-depleted alpha-lactalbumin (apo alpha-LA). This interaction leads to the formation of different supramolecular structures depending on the temperature. Heterogeneous, amorphous aggregates are formed at 5 degrees C, while droplets, coacervate-like structures, exist at 45 degrees C. The coacervates are formed by equimolar quantities of the two proteins, but their size and number depend on the initial protein molar ratio. These supramolecular structures are found to be stable when the temperature is decreased to 5 degrees C, while prolonged heating at 45 degrees C induces the formation of larger coacervates through a coalescence phenomenon. Surprisingly, interplay occurs between aggregates and coacervates when the temperature is increased from 5 to 45 degrees C. We discuss the results in terms of subtle heat-induced conformational changes in apo alpha-LA. In conclusion, our results show an association between globular proteins that leads to the formation of a variety of supramolecular structures in a temperature-dependent manner and confirm the primordial role of certain alpha-lactalbumin unfolding intermediates in protein-driven assembly.