Affinity Modification Research Articles

DNA duplexes containing 2'-deoxy-2'-iodoacetamidouridine as reagents for affinity modification of proteins

DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.

AFFINITY MODIFICATION OF EcoRII DNA METHYLTRANSFERASE BY THE DIALDEHYDE-SUBSTITUTED DNA DUPLEXES: MAPPING THE ENZYME REGION THAT INTERACTS WITH DNA

ABSTRACT Affinity modification of EcoRII DNA methyltransferase (M·EcoRII) by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine (Crib*) or 1-(β-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M·EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly268-Met391 of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M·EcoRII region. Our results support the theoretically predicted DNA binding region of M·EcoRII.

Analysis of the Antigen Combining Site: Correlations Between Length and Sequence Composition of the Hypervariable Loops and the Nature of the Antigen

It has long been suggested that the overall shape of the antigen combining site (ACS) of antibodies is correlated with the nature of the antigen. For example, deep pockets are characteristic of antibodies that bind haptens, grooves indicate peptide binders, while antibodies that bind to proteins have relatively flat combining sites. In 1996, MacCallum, Martin and Thornton used a fractal shape descriptor and showed a strong correlation of the shape of the binding region with the general nature of the antigen. However, the shape of the ACS is determined primarily by the lengths of the six complementarity-determining regions (CDRs). Here, we make a direct correlation between the lengths of the CDRs and the nature of the antigen. In addition, we show significant differences in the residue composition of the CDRs of antibodies that bind to different antigen classes. As well as helping us to understand the process of antigen recognition, autoimmune disease and cross-reactivity, these results are of direct application in the design of antibody phage libraries and modification of affinity.

Synergistic inhibitory activities of interleukin-10 and dexamethasone on human CD4+ T cells.

We have previously demonstrated that interleukin (IL)-10 synergizes with dexamethasone (Dex) in inhibiting proliferation of human T cells, stimulated in an antigen-presenting cell (APC)-dependent manner. Because IL-10 effectively inhibits APC accessory functions, the synergism could have been a result of its effect on APC. We then investigated the effects of Dex and IL-10 on T-cell subpopulations, stimulated in an APC-independent manner. CD4 and CD8 T cells were stimulated with anti-CD3, with or without Dex and IL-10, alone or in combination. Proliferation, glucocorticoid (GC) receptor binding, anti-CD3-induced tyrosine phosphorylation, IL-2 production, and expression of IL-2 receptor alpha, beta, and gamma chains were evaluated. The pharmacologic interactions were analyzed using the isobole method. IL-10 synergized with Dex in inhibiting CD4 but not CD8 T-cell proliferation. The synergism was not associated with modifications of GC receptor number or affinity, nor with modifications of anti-CD3-induced tyrosine phosphorylation. IL-10 synergized with Dex in inhibiting IL-2 production and increased Dex inhibitory effect on the expression of the IL-2 receptor alpha chain, which is up-regulated by CD3 stimulation and IL-2. Only Dex inhibited the beta and gamma chain expression, which, interestingly, is not up-regulated by IL-2. IL-2, as well as IL-7 and IL-15, reversed the effects of IL-10 but not those of Dex. IL-10 synergizes with Dex in inhibiting CD4 T-cell proliferation. Its synergizing effect is mediated by the inhibition of IL-2 production. Dex exerts additional activities, such as the inhibition of beta and gamma chain expression. Therefore, IL-10 could be useful for the enhancement of GC-based immunosuppressive therapies.

Pancreatic beta-cell alpha2A adrenoceptor and phospholipid changes in hyperlipidemic rats.

We previously showed that a 48-h intravenous lipid infusion in rats induces pancreatic beta-cell hypersensitivity to catecholamines. Our aim was to study the lipid-related changes that may account for such hypersensitivity in pancreatic islets. We show here that a 48-h increase in plasma FFA alters the binding characteristics of beta-cell alpha2 adrenoceptors in rats. Lipid infusion decreases pancreatic norepinephrine (NE) turnover rate by 28%, reflecting a reduction of pancreatic NE stores. Following lipid infusion, the density of alpha2 adrenoceptor binding sites is significantly lower and receptor affinity higher, both in islet homogenates (by three- and fivefold, respectively) and isolated whole beta-cells (by two- and sixfold, respectively). These changes correlate with the elevated insulin response to glucose found in lipid-infused rats. We also found a modification of islet phospholipid content, particularly in phosphoethanolamine species containing infused FA such as palmitate, oleate, stearate, and linoleate. This may account for the modifications in receptor affinity. These results suggest that hyperlipidemia-associated pathologies such as diabetes and obesity not only may result from alterations of metabolic pathways but also may be a consequence of early modifications in nervous firing rates and signal transduction pathways.

A spectroscopic study of the negative electron affinity of cesium oxide-coated diamond (111) and theoretical calculation of the surface density-of-states on oxygenated diamond (111)

The modification of the electron affinity of clean and oxygenated C(111) with cesium has been studied using ultra-violet photoelectron spectroscopy. Oxygenated C(111) shows strong valence emission features at 4.2 eV attributable to CO bonding orbital. Adsorption of cesium on the oxygenated diamond results in the formation of cesium oxide features at 4.2 eV and 8 eV and the condition of negative electron affinity (NEA). The cesium oxide adlayer is thermally stable to 500 °C and the NEA condition is not removed even at saturated O exposures, suggesting that cesium oxide-modified C(111) may act as a good photo-cathode. In order to evaluate the effect of oxygen atom adsorption on the surface states of C(111) 2×1 surface, the density of states of the O:C(111) surface adopting two different oxygen binding configurations have been calculated using the periodic density functional theory. The surface gap states on clean C(111) 2×1 surface are passivated by the adsorption of 1/2 monolayer oxygen in a bridging ‘epoxy’ fashion (C–O–C) across the carbon in the Pandey chain. At 1 monolayer oxygen coverage, the 2×1 reconstruction is lifted and an ‘on-top’ carbonyl (CO) binding mode results. Layered-resolved partial DOS calculation reveals that the surface band gap is closed with the adoption of the CO bonding configuration due to the introduction of quasi-continuous surface band states.

Investigation of surface oligonucleotide-binding proteins of eucaryotic cells by affinity modification with reactive oligonucleotide derivatives

Interactions between oligodeoxyribonucleotides (ODN) with different sequences and cell proteins were examined using the affinity modification by [32P]-labeled reactive oligonucleotide derivatives. 3"-Terminal ribouridine oxidized with sodium periodate, 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine, and the maleimide residue were used as reactive groups. All the compounds used are specific reagents. The set of the discovered nucleic acid-binding (NA-binding) proteins depends on the chemical properties of the affinity reagent. The presence of the hydrophobic group at the 5"-terminus of the ODN molecule is the key factor determining the variety of the discovered NA-binding proteins. The cells of different origin (A431, HeLa, KB, MCF-7, Hap-2, K562, Cos-7, NIH/3T3, human-lung primary epithelial cells, and porcine kidney primary cells) are characterized by the same set of NA-binding proteins whose affinity modifications depends on the conditions of incubation of oligonucleotides with the cells. Treatments of cells disturbing the integrity of the cellular membrane (scrapping, treatment with trypsin, or cell permeabilization with streptolysin O or saponin), disrupt interactions between NA-binding proteins from native cells and ODN.

Structural-functional analysis of bacteriophage T7 RNA polymerase.

This review summarizes our results of the structural and functional studies of bacteriophage T7 DNA-dependent RNA polymerase (T7 RNAP). Particular features of this enzyme (the single-subunit composition, relatively low molecular weight) make it the most convenient model for investigating the physicochemical aspects of transcription. The review discusses the main properties of T7 RNAP, interaction between the enzyme and promoter, principle stages of T7-transcription, and also the results of structural and functional studies by affinity modification and both random and site-directed mutagenesis techniques.

Identification of functional alpha-adrenoceptor subtypes in the bovine female genital tract during different phases of the oestrous cycle.

The concentration and functionality of the alpha-adrenoceptor (alpha-AR) subtypes in the genital tract of cyclic heifers were investigated. In each tissue sample, a single class of alpha1-ARs was observed, whereas two distinct classes of alpha2-ARs were discriminated: low-affinity (LA) and high-affinity (HA) alpha2-ARs. Statistical analysis showed the presence of significantly (p < 0.05) higher concentrations of all alpha-AR subtypes in the follicle than in the corpus luteum. No significant differences were found in the ovary or myometrium between the luteal and follicular phases. In the ovary, the density of alpha1-ARs was significantly (p < 0.05) higher than that of alpha2-ARs. By contrast, there were significantly (p < 0.05) more alpha2-ARs than alpha1-ARs in the myometrium. As far as alpha2-ARs are concerned, LA alpha2-ARs were significantly (p<0.05) higher than HA alpha2-ARs in all tested tissues. Competition studies suggested that the rank order of potency of antagonists for alpha1-ARs was prazosin > phentolamine > yohimbine, whereas for alpha2-ARs the order of potency was yohimbine > or = phentolamine>prazosin. Functional assays performed on myometrium showed that noradrenaline, phenylephrine and clonidine elicited concentration-dependent contractions only in dioestrus and pro-oestrus preparations and that clonidine was more effective than phenylephrine as a contractile agent. It appeared that there were no significant modifications in alpha-AR affinity or concentration during the different stages of bovine oestrous cycle, whereas the uterine spontaneous activity and the responsiveness to alpha-adrenergic stimulation was strongly influenced by hormonal levels. The modifications of uterine contractility observed during the oestrous cycle may be related to modifications induced in the transductional mechanisms of alpha-ARs.

CD and Melting Curves Structural Studies of the Tandem DNA Complex Formed with Oligonucleotides Carrying Photoactive and Sensitizing Groups in the Nick Region

Photoactive derivatives of oligonucleotides are widely used as affinity reagents for the study of structures and functions of nucleic acids and proteins. Between them the binary reagents are the more attractive in the last time. They represent the tandem of two oligonucleotide derivatives complementary to a target sequence and carrying photoactive and sensitizing groups. The efficiency of target modification in this case depends on the mutual arrangement in the nick region of photoactive and sensitizing groups, attached to the oligonucleotides. The use of binary reagents in affinity modification permits to reach the high selectivity of the process. In this work we report our studies on the thermodynamic and structural peculiarities of complementary tandem complex between DNA target and binary oligonucleotide reagent. The complex consisted of the target d(TTGAAGGGGACCGC) and two 7-mer oligonucleotide conjugates, one of which was modified on its 3′-phosphate with a photoreactive p-azidote-trafluorobenzaldehydehydrazone-group, and the other one was linked through its 5′-phosphate to a sensitizing perylene-group. Optical melting curves and thermal changes in circular dichroism (CD) spectra were detected for all possible oligonucleotide and/or conjugate combinations. In addition, molecular modeling simulation of the complex structure was carried out. It was found that CD spectra did not show serious changes in the B-helix structure of the duplex. The interaction between perylene- and azido-groups at the oligonucleotide junction led to considerable increase in duplex stability. CD and molecular modeling data clearly indicated that perylene-group interacted with the duplex in an intercalative manner, but azido-group located on the side of DNA chain minor groove.

Viral protein functions study by affinity modification.

The knowledge of virus reproduction is necessary to design new safe drugs for inhibition of infections. Ultra-violet irradiation of virus proteins with labeled virus genome fragments permits to identify specific nucleic acid binding proteins. Affinity modification of enzymes with nucleotide derivatives could help to determine NTP-binding proteins and those involved in viral genome replication. Photoreactive analogues of nucleic acids are among the tools used to detect elongation subunits of replicative complexes. Affinity modification approach has already resulted in successful treatment of virus diseases.

INTERACTION OF LNA OLIGONUCLEOTIDES WITH MDR1 PROMOTER

LNA oligonucleotides [1] can be used for targetting to double stranded DNA by the “strand invasion” mechanism. We used affinity modification by reactive oligonucleotide conjugates for investigation of oligonucleotides interaction with structured DNA. The tested LNAs and oligonucleotides of the same sequence were assayed as anti-mdr1 drugs in different cell cultures. One of the oligos, LNA79 strongly inhibited mdr1 induction in Hela cells and totally prevented activation of mdr1 in K-562.

The Heme–Globin and Dimerization Equilibria of Recombinant Human Hemoglobins Carrying Site-Specific β Chains Mutations

The heme–globin and dimer–tetramer equilibria of ferric recombinant human hemoglobins with site-specific β chain mutations at the heme pocket or at either the α1β1 or the α1β2 interfaces have been determined. The heme pocket mutation V67T leads to a marked stabilization of the β chain heme and does not affect the dimer–tetramer association constant, K2,4. In the C112 mutants, the intrinsic rate of β chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the α chains. Gel filtration experiments indicate that the K2,4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Substitution of cysteine 93 with A or M leads to a slight decrease of the rate of β chain heme release, whereas the obvserved K2,4 value is similar to that obtained for HbA-wt. Modifications in oxygen affinity were observed in all the mutant hemoglobins with the exception of V67T, C93A, and C112G. The data indicate that there is no correlation between tetramer stability, β chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.

Modulation of taurine uptake in the goldfish retina and axonal transport to the tectum. Effect of crushing the optic nerve or axotomy.

Although there are a great number of studies concerning the uptake of taurine in several tissues, the regulation of taurine transport has not been studied in the retina after lesioning the optic nerve. In the present study, isolated retinal cells of the goldfish retina were used either immediatly after cell suspension or in culture. The high-affinity transport system of [3H]taurine in these cells was sodium-, temperature- and energy-dependent, and was inhibited by hypotaurine and beta-alanine, but not by gamma-aminobutyric acid. There was a decrease in the maximal velocity (Vmax) without modifications in the substrate affinity (Km) after optic axotomy. These changes were mantained for up to 15 days after the lesion. The results might be the summation of mechanisms for providing extracellular taurine to be taken up by other retinal cells or eye structures, or regulation by the substrate taurine, which increases after lesioning the optic nerve. The in vivo accumulation of [3H]taurine in the retina after intraocular injection of [3H]taurine was affected by crushing the optic nerve or by axotomy. A progressive retinal decrease in taurine transport was observed after crushing the optic nerve, starting at 7 hours after surgery on the nerve. The uptake of [3H]taurine by the tectum was compensated in the animals that were subjected to crushing of the optic nerve, since the concentration of [3H]taurine was only different from the control value 24 hours after the lesion, indicating an efficient transport by the remaining axons. On the contrary, the low levels of [3H]taurine in the tectum after axotomy might be an index of the non-axonal origin of taurine in the tectum. Axonal transport was illustrated by the differential presence of [3H]taurine in the intact or crushed optic nerve. The uptake of [3H]taurine into retinal cells in culture in the absence or in the presence of taurine might indicate the existence of an adaptive regulation of taurine transport in this tissue, however taurine transport probably differentially occurs in specific populations of retinal cells. The use of a purified preparation of cells might be useful for future studies on the modulation of taurine transport by taurine in the retina and its role during regeneration.

A lysine residue involved in the inhibition of vacuolar H(+)-pyrophosphatase by fluorescein 5'-isothiocyanate.

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a central role in the electrogenic translocation of protons from cytosol to the vacuole lumen at the expense of PP(i) hydrolysis. A fluorescent probe, fluorescein 5'-isothiocyanate (FITC), was used to modify a lysine residue of vacuolar H(+)-PPase. The enzymatic activity and its associated H(+) translocation of vacuolar H(+)-PPase were markedly decreased by FITC in a concentration-dependent manner. The inhibition of enzymatic activity followed pseudo-first-order rate kinetics. A double-logarithmic plot of the apparent reaction rate constant against FITC concentration yielded a straight line with a slope of 0.89, suggesting that the alteration of a single lysine residue on the enzyme is sufficient to inhibit vacuolar H(+)-PPase. Changes in K(m) but not V(max) values of vacuolar H(+)-PPase as inhibited by FITC were obtained, indicating that the labeling caused a modification in affinity of the enzyme to its substrate. FITC inhibition of vacuolar H(+)-PPase could be protected by its physiological substrate, Mg(2+)-PP(i). These results indicate that FITC might specifically compete with the substrate at the active site and the FITC-labeled lysine residue locates probably in or near the catalytic domain of the enzyme. The enhancement of fluorescence intensity and the blue shift of the emission maximum of FITC after modification of vacuolar H(+)-PPase suggest that the FITC-labeled lysine residue is located in a relatively hydrophobic region.

Analog of mRNA, pUUUGUU derivative with an arylazide group at guanosine residue, crosslinks with nucleotides A1823 and A1824 of 18S rRNA in human 80S ribosomes

Affinity modification of human placental 80S ribosomes was performed using an mRNA analog, pUUUGUU derivative containing a 3′-O-methyl group and photoactivatedp-azidotetrafluorobenzoyl group at the N7 atom of guanine, in complexes with one or two codon-anticodon interactions formed with participation of Phe-tRNAPhe or Phe-tRNAPhe and Val-tRNAVal (in both cases, the modified codon GUU was at the A site). It was established by the reverse transcription technique that the mRNA analog crosslinks with nucleotides A1823 and A1824 of 18S rRNA in the complexes of both types.

A direct photo-activated affinity modification of tetracycline transcription repressor protein TetR(D) with tetracycline

Results of a first successful application of a direct photo-induced affinity modification of Tet repressor (TetR(D)) protein with tetracycline within a complex of known three-dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR(D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59–Glu73 and Ala173–Glu183, which form a binding tunnel for the drug according to the X-ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.

Periodate oxidation in chemistry of nucleic acids: Dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides (Review)

The employment of periodate oxidation in the chemistry of nucleic acids and their components is reviewed. The reaction mechanism, structural requirements to substrates, and synthesis of dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides are discussed in the first part. The second part involves chemical, physicochemical, and biological properties of the dialdehyde derivatives, as well as their use for the affinity modifications of proteins.

Modulation of dimerization, binding, stability, and folding by mutation of the neurophysin subunit interface.

Bovine neurophysins, which have typically served as the paradigm for neurophysin behavior, are metastable in their disulfide-paired folded state and require ligand stabilization for efficient folding from the reduced state. Studies of unliganded porcine neurophysin (oxytocin-associated class) demonstrated that its dimerization constant is more than 90-fold greater than that of the corresponding bovine protein at neutral pH and showed that the increased dimerization constant is accompanied by an increase in stability sufficient to allow efficient folding of the reduced protein in the absence of ligand peptide. Using site-specific mutagenesis of the bovine protein and expression in Escherichia coli, the functional differences between the bovine and porcine proteins were shown to be attributable solely to two subunit interface mutations in the porcine protein, His to Arg at position 80 and Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relatively small impact on dimerization, while mutation to either Glu or Asp markedly reduced dimerization in the unliganded state, albeit with apparent retention of the positive linkage between dimerization and binding. Comparison of the peptide-binding constants of the different mutants additionally indicated that substitution of His-80 led to modifications in binding affinity and specificity that were independent of effects on dimerization. The results demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contribution of the energetics of ligand-induced conformational change in this region to the overall thermodynamics of binding. The potential utility to future studies of the self-folding and monomeric mutants generated by altering the interface is noted.

Concordant decreases of thyroxine and thyroxine binding protein concentrations during sepsis

The principal thyroxine (T4) binding proteins were measured in 8 septic patients and 8 controls to determine the extent to which a decrease in their concentration contributes to the decrease in serum T4 in sepsis. T4 binding globulin (TBG) evaluated by radioimmunoassay (RIA) and radial immunodiffusion (RID) was 61% and 66%, respectively, of the normal mean value in sera from septic patients. Decreases of albumin and transthyretin (TTR) to 55% and 29%, respectively, of the normal mean concentration contributed to the loss of T4 binding power in these sera. Total serum binding of T4, calculated from the normal contribution of each protein to T4 binding and the reduction of its concentration in septic patients, was 55% of that in the normal controls. This decrease in the estimated protein binding of T4 was proportional to the decrease of serum T4 from a mean of 8.4 microg/dL in the normal controls to 4.7 microg/dL (56% of the control) in the septic patients. Because TBG binds most of the serum T4, the decrease in the TBG concentration was the major factor in the decrease of total T4 binding power. Since the concentration of the major T4 binding proteins decreased sufficiently to account for the decrease in serum T4, it is unnecessary to postulate the effects of additional factors such as binding inhibitors or modification of T4 binding protein affinity.