Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.

Putative receptor binding sites on alphaviruses as visualized by cryoelectron microscopy.

The structures of Sindbis virus and Ross River virus complexed with Fab fragments from monoclonal antibodies have been determined from cryoelectron micrographs. Both antibodies chosen for this study bind to regions of the virions that have been implicated in cell-receptor recognition and recognize epitopes on the E2 glycoprotein. The two structures show that the Fab fragments bind to the outermost tip of the trimeric envelope spike protein. Hence, the same region of both the Sindbis virus and Ross River virus envelope spike is composed of E2 and is involved in recognition of the cellular receptor.

Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.

High-affinity binding sites for histone H1 in plasmid DNA.

The interaction of histone H1 isolated from chicken erythrocytes with restriction fragments from plasmids pBR322 and pUC19 was studied by gel electrophoresis. Certain restriction fragments exhibited unusually high affinity for the histone, forming high molecular mass complexes at protein to DNA ratios at which the other fragments did not show evidence for binding. The highly preferred fragments are intrinsically curved, as judged by their electrophoretic mobility in polyacrylamide gels, by computer modeling, and by imaging with scanning force microscopy. However, control experiments with either curved portions of the same fragments or highly curved kinetoplast DNA fragments showed that the presence of curvature alone was not sufficient for preferential binding. By using various restriction fragments centered around the highly preferred sequence, it was found that the high-affinity binding required in addition the presence of specific sequences on both sides of the region of curvature. Thus, both curvature and the presence of specific sites seem to be required to generate high affinity.

Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.

Localization of specific erythropoietin binding sites in defined areas of the mouse brain.

The main physiological regulator of erythropoiesis is the hematopoietic growth factor erythropoietin (EPO), which is induced in response to hypoxia. Binding of EPO to the EPO receptor (EPO-R), a member of the cytokine receptor superfamily, controls the terminal maturation of red blood cells. So far, EPO has been reported to act mainly on erythroid precursor cells. However, we have detected mRNA encoding both EPO and EPO-R in mouse brain by reverse transcription-PCR. Exposure to 0.1% carbon monoxide, a procedure that causes functional anemia, resulted in a 20-fold increase of EPO mRNA in mouse brain as quantified by competitive reverse transcription-PCR, whereas the EPO-R mRNA level was not influenced by hypoxia. Binding studies on mouse brain sections revealed defined binding sites for radioiodinated EPO in distinct brain areas. The specificity of EPO binding was assessed by homologous competition with an excess of unlabeled EPO and by using two monoclonal antibodies against human EPO, one inhibitory and the other noninhibitory for binding of EPO to EPO-R. Major EPO binding sites were observed in the hippocampus, capsula interna, cortex, and midbrain areas. Functional expression of the EPO-R and hypoxic upregulation of EPO suggest a role of EPO in the brain.

Assessing computational tools for the discovery of transcription factor binding sites

The prediction of regulatory elements is a problem where computational methods offer great hope. Over the past few years, numerous tools have become available for this task. The purpose of the current assessment is twofold: to provide some guidance to users regarding the accuracy of currently available tools in various settings, and to provide a benchmark of data sets for assessing future tools.

Behavioural and neurochemical correlates of drugs acting at imidazoline and a2-adrenoceptor binding sites

A number of agents with differing selectivity profiles for the non-a2 adrenoceptor binding site (NAIBS), imidazoline preferring receptor (IPR) and a2-adrenoceptor were employed in a series of behavioural and neurochemical experiments to determine a functional role for the former two sites. The highly selective NAIBS ligand RX801 077 produced an increase in rat brain extracellular noradrenaline (NA) levels, as determined by the technique of in vivo microdialysis, which may underlie its ability to produce a discriminable cue in the same species. This increase in NA may be due to a suggested link between the NAIBS and the monoamine oxidase inhibitor (MAOI) activity of RX801 077. For instance, the RX801 077 cue was substituted for by the MAOI drugs pargyline and moclobemide, which themselves down regulate NAIBS when administered chronically. RX811 059 substituted for the RX801 077 cue which may be due its ability to stimulate NA release via its activity as a highly selective a2-adrenoceptor antagonist. An effect upon NA output may also explain the ability of RX801 077 to 'mimic' the anti-immobility effect of the antidepressant drug desmethylimipramine (DMJ) in the forced swimming test. Further studies are therefore required to examine a possible role for the NAIBS in the treatment of depression. Discriminable cues were also produced by RX811 059 and the a2- adrenoceptor agonist clonidine, probably as a consequence of their respective ability to stimulate and inhibit NA output via their opposing activity at a2-adrenoceptors. The IPR has been suggested to play a role in mediating the hypotensive effect of clonidine, although a precise role was unable to be established for this site in the present studies due to the unavailability of highly selective IPA agents.

Identification of putative steroid-binding sites in human ABCB1 and ABCG2

Homology modelling was used to generate three-dimensional structures of the nucleotide-binding domains (NBDs) of human ABCB1 and ABCG2. Interactions between a series of steroidal ligands and transporter NBDs were investigated using an in silico docking approach. C-terminal ABCB1 NBD (ABCB1 NBD2) was predicted to bind steroids within a cavity formed partly by the P-Loop, Tyr1044 and Ile1050. The P-Loop within ABCG2 NBD was also predicted to be involved in steroid binding. No overlap between ATP- and RU-486-binding sites was predicted in either NBD, though overlaps between ATP- and steroid-binding sites were predicted in the vicinity of the P-Loop in both nucleotide-binding domains.

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to ets binding sites

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg2+, Na+, K+ and NH4 (+) and inhibition by ouabain of (Na+,K+)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, . (Na+,K+)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP ( (M) = 0.09 +/- A 0.01 mmol L-1) of the decapodid III (Na+,K+)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na+,K+)-ATPase activity by K+ also revealed a threefold greater affinity for K+ ( (0.5) = 0.91 +/- A 0.04 mmol L-1) in decapodid III than in other stages; NH4 (+) had no modulatory effect. The affinity for Na+ ( (0.5) = 13.2 +/- A 0.6 mmol L-1) of zoea I (Na+,K+)-ATPase was fourfold less than other stages. Modulation by Na+, Mg2+ and NH4 (+) obeyed cooperative kinetics, while K+ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg2+ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg2+-stimulated ATPases other than (Na+,K+)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na+-ATPase may be involved in the ontogeny of osmoregulation in larval The NH4 (+)-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Sequences of immunoglobulin chains : tabulation and analysis of amino acid sequences of precursors, V-regions, C-regions, J-chain and -microglobulins /

1976 ed. issued under title: Variable regions of immunoglobulin chains.

Effect of terbium(III) on the binding of aromatic guests with sodium taurocholate aggregates

The effect of binding Tb(3+) to sodium taurocholate aggregates containing polyaromatic hydrocarbon guests was examined using pyrene and 1-ethylnaphthalene as guests that bind to the primary aggregate, and 1-naphthyl-1-ethanol as a secondary aggregate guest. Time-resolved fluorescence quenching studies were used to study the binding site properties, while laser flash photolysis quenching studies provided information on the dynamics of the guest-aggregate system. Both the primary and secondary aggregate binding sites became more compact in the presence of bound Tb(3+), while only the primary aggregate became more accessible to anionic molecules. The binding dynamics for the guest-primary aggregate system became faster when Tb(3+) was bound to the aggregate. In contrast, for the guest-secondary aggregate the presence of Tb(3+) resulted in a small decrease in the dissociation rate constant. The influence of bound Tb(3+) on the primary and secondary bile salt aggregates is significantly different, which affects how these aggregates can be used as supramolecular host systems to modify guest reactivity.