Original Binding Site Research Articles

Quantitative characterization of the binding of fluorescently labeled colchicine to tubulin in vitro using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a new technique that allows the determination of the diffusion constant of a fluorescent molecule in solution. Also, the binding of the fluorescent molecule to a target can be analyzed, if the difference in the diffusion coefficients of the free and bound ligand is sufficiently large. With FCS, the interaction between fluorescein-colchicine (FC) and tubulin has been studied in vitro. A fast and reversible binding is observed with an association constant at room temperature of (3.9 +/- 0.1) x 10(4) M-1. No competition with colchicine is seen, indicating that FCS reveals the existence of a new binding site on tubulin. FCS is not able to show the binding of FC to the original colchicine binding site, even though it exists, because the fluorescence of FC is strongly quenched upon binding to this site. This quenching is evident in spectrofluorometry experiments, revealing a slow binding of FC to tubulin that is subject to competition with colchicine. FCS allows the determination of the diffusion coefficients of both free and bound fluorescent colchicine which were found to be (2.6 +/- 0.2) x 10(-)10 and (2.0 +/- 0.2) x 10(-)11 m2 s-1, respectively. It can be concluded that fluorescent labeling, especially of small molecules, can interfere considerably with the binding behavior that is being studied. Although general qualitative effects in vivo are similar for colchicine and its fluorescein derivative, this quantitative study of the binding to tubulin presents a nuanced view, and the existence of a second binding site for FC can even explain some conflicting indications in the literature.

Spin-Polarized Radical Pair in Photosystem I Reaction Center That Contains Different Quinones and Fluorenones as the Secondary Electron Acceptor

The spin-polarized radical pair of the primary donor chlorophyll P700+ and the secondary acceptor Q- was studied by the electron spin echo envelope modulation (ESEEM) technique in the spinach photosystem I reaction center in which artificial quinones and fluorenones were reconstituted as Q. From the dipole interaction between the spins on P700+ and Q-, the distance between P700 and anilinochloronaphthoquinone or hydroxyanthraquinone was estimated to be 25.6 ± 0.3 Å or 25.7 ± 0.3 Å, respectively, suggesting their proper binding at the original phylloquinone binding site. Distances of 26.0 Å with larger heterogeneity were estimated for (NO2)3- and (NO2)4-fluorenone compounds that have only one carbonyl group, suggesting their multiple orientations in the binding pocket. The lifetimes of the radical pairs estimated by the time dependency of the two-pulse ESE signals were almost temperature independent (30−80 μs at 4−80 K). In the three-pulse ESE measurement, the lifetimes were lengthened by 10−103 times and showed strong temperature dependencies.

Structural Determinants Present in the C-terminal Binding Protein Binding Site of Adenovirus Early Region 1A Proteins

The C-terminal binding protein (CtBP) has previously been shown to bind to a highly conserved six-amino acid motif very close to the C terminus of adenovirus early region 1A (Ad E1A) proteins. We have developed an enzyme-linked immunosorbent assay that has facilitated the screening of synthetic peptides identical or similar to the binding site on Ad E1A for their ability to bind CtBP and thus inhibit its interaction with Ad12 E1A. It has been shown that amino acids both C-terminal and N-terminal to the original proposed binding site contribute to the interaction of peptides with CtBP. Single amino acid substitutions across the binding site appreciably alter the Kd of the peptide for CtBP, indicative of a marked reduction in the affinity of the peptide for CtBP. The solution structures of synthetic peptides equivalent to the C termini of both Ad5 and Ad12 E1A and two substituted forms of these have been determined by proton NMR spectroscopy. Both the Ad12 and Ad5 peptides dissolved in trifluoroethanol/water mixtures were found to adopt regular secondary structural conformations seen as a series of beta-turns. An Ad12 peptide bearing a substitution that resulted in only very weak binding to CtBP (Ad12 L258G) was found to be random coil in solution. However, a second mutant (Ad12 V256K), which bound to CtBP rather more strongly (although not as well as the wild type), adopted a conformation similar to that of the wild type. We conclude that secondary structure (beta-turns) and an appropriate series of amino acid side chains are necessary for recognition by CtBP.

Proton rotating frame spin-lattice relaxation study of slow motion of pore water

Proton rotating frame spin-lattice relaxation times of water in hydrated porous silica glass, with pore diameters of 7.4, 23.7, 49.1, and 54.8 nm, are reported. Experiments were limited to hydrations corresponding to more than one-third hydrated pore volumes. The frequency dependence of the relaxation rates reveals a slow motion of pore water with a characteristic correlation time of ∼10−5 s. Within the limits given above, this correlation time depends on pore dimension and hydration only slightly. This indicates that the surface water molecules, which reorient with a characteristic time of ∼10−8 s, are changing their orientational axis with the slower correlation time ∼10−5 s. This process, which disrupts the nearby water molecules, depends primarily on the environment in the vicinity of the molecule’s original binding site. The analysis, based on the two-site fast exchange model, indicates that in small pores all the water may be affected by these slow jumps.

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.

Interactions between the finger and kringle-2 domains of tissue-type plasminogen activator and plasminogen activator inhibitor-1.

We have shown that plasminogen activator inhibitor-1 (PAI-1) inhibits the fibrin binding of both the single chain and two chain forms of tissue-type plasminogen activator (tPA) through two different mechanisms. PAI-1 inhibits the finger domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated single chain tPA and the kringle-2 domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated two chain tPA. In accordance with the data, preformed complexes of single chain tPA/PAI-1 and of two chain tPA/PAI-1 lost the fibrin binding abilities mediated by the finger and kringle-2 domains, respectively. These effects of PAI-1 appear to be mediated by steric hindrance of the fibrin binding sites after PAI-1 binding to adjacent regions in the functional domains of tPA. We thus propose a model in which a PAI-1 binding site resides in the finger domain of a single chain, and plays a role in the reversible association of single chain tPA and PAI-1. Conformational changes may take place during the conversion of single chain tPA to two chain tPA, resulting in burying of the original PAI-1 binding site and exposure of an alternate PAI-1 binding site on the surface of the kringle-2 domain.

Extension of the DNA binding consensus of the chicken c-Myb and v-Myb proteins.

The chicken c-myb gene and the v-myb oncogene transduced by avian myeloblastosis virus (AMV) encode DNA binding transcription activators. The DNA binding domain of AMV v-Myb displays a number of amino acid changes relative to c-Myb; v-Myb proteins in which one or more of three crucial residues in the DNA binding domain are mutated to resemble the c-Myb sequence display altered transformation phenotypes. In order to establish whether the spectrum of DNA binding sites which AMV v-Myb can recognise is different from that seen by chicken c-Myb, a site selection protocol was used to derive consensus binding sequences for three variant Myb proteins made in vitro, and also using nuclear extract from the v-myb transformed cell line BM2. The results show that the original consensus binding site defined for v-Myb, YAA-CKG, can be extended to YAACKGHH, and that this new consensus holds for both v-Myb and chicken c-Myb.

Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer

We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within the polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, alpha-1 to alpha-4 and beta-1 to beta-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides possessed DNA-binding activities with exactly the same sequence specificity. Individual alpha or beta polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when alpha and beta polypeptides were mixed together in any arbitrary pair. These observation along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of alpha and beta subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2.

Binding characteristics of [ 3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [ 3H]peptidergic opioi ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [ 3H]-[ d-Ala 2-, Met 5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl 2 and 100 mM NaCl at 0 °C (on ice) for 3 h. With this assay condition, the significant binding of followint [ 3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [ 3H]-[ d-A;a 2, MePhe 4, Gly-ol 5]enkephalin (μ-type), [ 3H]-[ d-Ala 2, d-Leu 5]enkephalin (δ-type) and [ 3H]dynorphin 1–9 ( K-type). The number of binding sites in solubilized preparations for each [ 3H]ligand corresponded to 40–50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [ 3H]ligand. Apparent dissociation constants between solubilized receptors and [ 3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycogdeoxycholate and NaCl and the dilution process.

A search for site-filling ligands in the Mcg bence-jones dimer: Crystal binding studies of fluorescent compounds

In trigonal crystals grown in 1.9 M ammonium sulfate buffered at pH 6.2, the Mcg light-chain (Bence-Jones) dimer has a highly aromatic binding cavity accessible to a wide range of hydrophobic and aromatic ligands. A search was made for site-filling ligands by diffusing compounds into the crystals and determining their locations, orientations and relative occupancies by difference Fourier analysis at 2.7-Å resolution. 1-Anilinonaphthalene-8-sulfonate, a small ligand in comparison with the rest of the series, initially occupied a site in the main binding cavity. With time, however, this ligand changed its position to the deep binding pocket beyond the floor of the main cavity. The original binding site remained vacant, despite the presence of a large excess of ligand in the soaking solution. Ligands increasing in size from fluorescein to bis( N-methyl)acridine (lucigenin) to dimers of carboxytetramethylrhodamine were found to bind with stringent stereospecificity in the main cavity, but the mode of binding was different in each case. The dimer of the 6-isomer of carboxytetramethylrhodamine, in which the two carboxyl groups are in para positions on the phenyl moiety, proved to be an effective site-filling ligand. The differences in the binding properties of dimers of 5- and 6-carboxytetramethylrhodamine led to an explanation for isomeric discrimination in the binding site. There were extensive conformational changes in the binding cavity to accommodate the ligands, particularly 6-carboxytetramethylrhodamine. The second and third hyper-variable loops proved very flexible, and moved in ways to expand the binding site. The side chains of key tyrosine and phenylalanine residues in the site were also highly mobile. Their orientations adjusted to optimize complementarity with the ligands. These conformational adjustments are consistent with the tenets of a limited neo-instructive theory of ligand binding.

Interaction of murine colony-stimulating factor (CSF-1) with alveolar mononuclear phagocytes

Colony-stimulating factor (CSF-1) was purified from serum-free L-cell-conditioned medium (LCM) and iodinated so that we could study its interaction with murine alveolar macrophages. At 0 °C, the binding of 125 ICSF-1 to alveolar macrophages reached a stable maximum within 16 h. Under this condition, the binding of 125 ICSF-1 at various concentrations was saturated at about 3 ng/ml. The binding sites of 125 ICSF-1 were sensitive to trypsin but not to DNase or RNase treatment. At 37 °C, the trypsin-treated cells regenerated more than 90% of their original binding sites within 12 h. Whereas more than 97% of these alveolar macrophages were phagocytic and esterase-positive, autoradiographic studies showed that only 10–31 % of them were capable of binding to 125 ICSF-1. These results indicate that the frequencies of CSF-1-binding cells and alveolar macrophage colony-forming cells (AL-CFC) are closely correlated, but no causal relationship has been established.

Structural studies on the chromophore attachment site on rhodopsin following bleaching

dopsin is accompanied by isomerisation of the chromophore 1 lcis retinal, and conformational changes in the polypeptide, opsin [ 11. The resultant effect is the conversion of electromagnetic energy into a neural signal. Long after the effective primary event has occurred, opsin continues to progress through a series of spectrally defined structures [2,3]. During this process, the now all-trans chromophore migrates from the original binding site [4]. Following our identification of this primary attachment site for 1 l-cis retinal [ 5,6], we investigated the process of migration. These studies indicate that the chromophore remains attached to the same lysyl residue for at least 5 min after illumination and that subsequent migration occurs to the lipid milieu rather than onto any further lysyl side-chains. mophore to any lysine with which it forms a Schiff base. The membranes were dissolved and subjected to SDS-urea polyacrylamide gel electrophoresis and 2 mm gel slices analysed for their radioactive content. The fragments generated by digestion of the protein with V8 protease were isolated from the washed membranes by gel filtration with Sephadex LH60 in formic acid (90%, v/v): acetic acid:chloroform: ethanol, 2:2:4:2 (v/v) [6]. Cleavage of the small V8 fragment (V8-S) was carried out in 70% formic acid for 24 h in the dark using 100.fold molar excess (with respect to methionines) of cyanogen bromide [6]. Further purification was carried out with Sephadex LH60 as above, only using formic acid (90%, v/v): ethanol as solvent. Sephadex LH60 was obtained from Pharmacia Fine Chemicals, S. aureus V8 protease from Miles Biochemicals and KBH4 from BDH Ltd.

42] Cyanoborohydride reduction of rhodopsin

Publisher Summary This chapter discusses the cyanoborohydride reduction of rhodopsin. The dark-adapted form of rhodopsin resists reduction, apparently because the binding site is deep in a hydrophobic pocket of the molecule, inaccessible to small hydrophilic molecules. This contrasts with the more accessible retinal binding sites of cone pigments, which can be directly reduced by sodium borohydride. Cyanoborohydride is a more hydrophobic borohydride derivative. It serves to penetrate into the hydrophobic core of the rhodopsin molecule and reduce the retinal linkage to lysine. The usefulness of the reaction, beyond confirming that the dark-adapted form of rhodopsin has a retinal Schiff base, is that it chemically binds the retinal to its original binding site in a secondary amino linkage that is not subject to hydrolysis or transaminization to other amino groups in the process of fragmentation and purification. Two additional advantages of cyanoborohydride are, first, that it has a greater specificity for Schiff base reduction relative to aldehyde reduction and, second, that it has a greater stability at neutral and acid pH values.

Attachment site(s) of retinal in bacteriorhodopsin.

After chemical reduction of the retinylidene-lysine Schiff base linkage in bacteriorhodopsin, the retinyl residue is covalently attached to Lys-216 (with a possible minor fraction on Lys-172) or to both Lys-216(172) and Lys-40/41. The linkage site (up to 100% on Lys-216; up to 70% on Lys-40/41) depends on whether the sample is reduced in the light or dark, whether the sample is light or dark adapted, and on temperature. Absorbance and circular dichroism spectra indicate that the retinyl residue is in its original binding site after reduction in the light. Thus, the different attachment sites may reflect changes that occur during the photoreaction cycle or during light/dark adaptation, or the reduction of accidental physiologically irrelevant Schiff base linkages to lysines close to the normal linkage in the structure of bacteriorhodopsin. In either case, the retinal does not leave its binding site. This last point severely limits the possible arrangements of the amino acid sequence in the bacteriorhodopsin tertiary structure and clearly distinguishes two models that are consistent with all criteria.

Enhancement of anion equilibrium exchange by dansylation of the red blood cell membrane.

Dansylation of resealed red cell ghosts enhances the band 3 protein-mediated equilibrium exchange of sulfate ions. After dansylation, the specific anion transport inhibitor 4,4'-diisothiocyanato-dihydrostilbene 2,2'-disulfonate (H2DIDS) is still capable of combining with its original binding site on the band 3 protein and of producing the same high degree of inhibition of sulfate exchange as in the untreated red cell ghost. Nevertheless, dansylation causes allosteric effects at the H2DIDS-binding site that exhibit themselves by an increased susceptibility to dinitrophenylation of one of the amino acid residues that is involved in the covalent bond formation with H2DIDS and a decrease of the apparent KI values for two reversibly acting inhibitors that are known to produce their effects at the H2DIDS-binding site of the band 3 protein. The degree of enhancement of divalent anion exchange depends on both the pH that existed during dansylation and the pH at which the anion equilibrium exchange across the dansylated membrane is measured. The effect of dansylation reaches a broad maximum around ph 6.6. In untreated ghosts, divalent anion equilibrium exchange passes through a maximum around pH 6.3. After dansylation under optimal conditions at pH 6.6, anion equilibrium exchange as measured below the maximum of pH 6.3 is much less enhanced than above the maximum. Under suitable experimental conditions, the maximum may be replaced by a plateau that extends up to at least pH 8.5. At this pH, the enhancement is about 100-fold. Thus, the pH dependence of divalent anion exchange becomes more similar to that of monovalent anion exchange. The apparent activation enthalpies for sulfate-equilibrium exchange across the modified membrane, as measured at pH 6.3 and 7.9, are indistinguishable, independent of temperature between 0 and 37 degrees C and amount to 146 kj/mol. This is similar to the activation enthalpies measured in the untreated membrane. The mode of action of dansyl chloride is discussed on the basis of currently considered mechanisms of divalent anion transport, for which the pertinent equations are presented.

The orientation of the chromophore of vertebrate rhodopsin in the “meta” intermediate states and the reversibility of the meta II-meta III transition

The “meta” states of rhodopsin have been studied by measuring the linear dichroism of magnetically oriented suspensions of rod outer segments isolated from frog and cattle retinae. In cattle rods, the absorbing dipole of metarhodopsin I (MI) has the same orientation with respect to the plane of the disc membrane as in rhodopsin. A rotation of the chromophore is observed in both frog and cattle rods during the formation of metarhodopsin II (MII). In frog rods this rotation is reversed upon the MII ı MIII step which is shown to be a pH dependant equilibrium. In MIII, retinal is still attached to the original protein binding site and gives rise to an induced circular dichroism signal. The simple decay scheme

Transient light-induced conformational changes in rhodopsin.

Arguments are presented which support the possibility that the unfolding of the rhodopsin molecule during photolysis up to the stage of metarhodopsin II is followed by a spontaneous refolding of the protein, once the isomerized retinaldehyde has left its original binding site. Such a transient conformational change might imply a very similar conformation for rhodopsin and opsin, apart from the presence of the chromophore.

Use of photoreceptor membrane suspensions for the study of rhodopsin and associated enzyme activities

Photoreceptor membrane preparation, enriched in rhodopsin content by treatment with 11- cis-retinaldehyde, offer several advantages over detergent-solubilized rhodopsin preparations for the study of the properties of the visual pigment. After a brief description of the chemical composition of the photoreceptor membrane and the role of its phospholipids, chemical modification of the membrane amino groups is reported, leading to the establishment of the chromophoric binding site in native rhodopsin. Through application of the reducing agent sodium borohydride it is demonstrated that the chromophore remains at its original binding site during the conversion to metarhodopsin II, whereas the decay of this substance leads to migration of the chromophore, In the absence of NADPH, which precludes reduction to retinol, 60% of the chromophore remains bound to amino groups of proteins and phospholipids in the membrane. Binding to an amino group in the active site of retinoldehydrogenase takes part in the migrational sequence. Chemical modification of sulfhydryl groups shows that in the absence of detergent no additional SH-groups are exposed upon illumination. This excludes a role of SH-groups in the binding of the chromophore. Proteolytic degradation by pronase of rhodopsin in membrane suspensions is possible without the loss of 500-nm absorbance, which occurs with detergent-solubilized rhodopsin.

Two forms of copper (II) in fungal laccase

1. 1. The nature of different forms of copper in fungal laccase ( p-diphenol:O 2 oxidoreductase, EC 1.10.3.2) and their possible role in the catalytic activity has been studied by electron paramagnetic resonance (EPR) and proton relaxation measurements. 2. 2. By comparisons between experimental and simulated EPR spectra at 9 and 35 GHz it is shown that both laccase A and B contain two forms of Cu 2+, each corresponding to 1 atom per mole of protein. One (represented by Signal 1) corresponds to the spectrally unique form described earlier and shows a small deviation from axial symmetry, while the other one (Signal 2) represents the form earlier attributed to denatured molecules. Data obtained here suggest that both forms are present in the same protein molecule. 3. 3. Proton relaxation measurements show that neither form binds easily exchangeable water. However, the form with Signal 2 gives the largest contribution to the enhancement, indicating that it is more available. This is supported by the finding that only this form is affected by various solutes. 4. 4. Denaturation of the enzyme with guanidine · HCl destroys Signal 1 as well as the characteristic absorption at 610 mμ. Treatment with azide, on the other hand, decreases Signal 2 without affecting the blue colour. 5. 5. The rates of reduction by quinol of Cu 2+ giving Signals 1 and 2, as well as of the corresponding forms in ceruloplasmin, and of added Cu 2+ have been measured. Signal 1 is reduced much more rapidly than Signal 2, but this still reacts considerably faster than added Cu 2+. 6. 6. Treatment of the enzyme with cyanide produces, with concomitant loss of oxidase activity, an entirely new EPR spectrum corresponding to 40% of the original intensity. The treatment is entirely reversible which indicates that Cu 2+ is still at its original binding site. The new spectrum suggests coordination of Cu 2+ to three to four N atoms.