Synthetic peptide mimicking of binding sites on olfactory receptor protein for use in 'electronic nose'

A putative tertiary structure model of the dog's olfactory receptor (olfd canfa) is established in this study. By using a target odorous compound (trimethylamine), it is possible to locate the most plausible binding sites between the receptor model structure and the target odorous molecules through computer docking simulations. The two short oligo-peptide sequences (orp61 and orp188) for trimethylamine sensing were identified, synthesized, purified and coated onto the surface of the separate piezoelectric gold electrodes. These two peptides show a high binding capability for trimethylamine. To further enhance the sensitivity of the polypeptides towards the target compound, the polarity and the degree of docking were changed by a site-specific modification technique. The orp61 sequence was modified by substituting two amino acids in the binding pocket resulting in 33% increase in sensitivity towards trimethylamine and reduced noises from other non-target chemicals. The techniques used in the present study offer a unique approach for synthesizing peptides in mimicking binding domain of olfactory receptors. The approach can be easily applied to further development of recognized molecules for gas sensing, especially for use in 'electronic noses'.     

Separate subunits for agonist and benzodiazepine binding in the gamma-aminobutyric acidA receptor oligomer

The gamma-aminobutyric acidA (GABAA) agonist muscimol can be photoactivated by 254 nm illumination to affinity label its binding site in the GABAA receptor. We have conducted this reaction on the pure receptor from bovine cerebral cortex in detergent solution, showing that [3H]muscimol can produce then a specific saturable labeling. In the detergent solution, the receptor alone is sensitive to 254 nm irradiation; this reduces the efficiency of incorporation to below that in the membranes, but the competing photoreaction with [3H]muscimol is sufficient and occurs at a representative set of the muscimol-binding sites, such that it can be employed for the photolabeling of those sites. The affinity of [3H]muscimol displayed in this irreversible reaction is indistinguishable from that of its reversible binding. gamma-Aminobutyric acid and bicuculline compete in the photolabeling reaction according to their known affinities at the gamma-aminobutyric acid-binding site. The labeling is shown to occur at the beta-subunit (apparent Mr 57,000) in the pure receptor. The binding sites for gamma-aminobutyric acid agonists, on the beta-subunits, and the benzodiazepine binding sites, on the alpha-subunits, are linked allosterically so that a strongly cooperative hetero-oligomeric structure of this receptor is deduced.     

Solubilization of convulsant/barbiturate binding activity on the gamma-aminobutyric acid/benzodiazepine receptor complex

Binding activity of the radioactive cage convulsant [35S]t-butylbicyclophosphorothionate was solubilized from rat brain membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio] propanesulfonate. Binding (KD = 26 nM, Bmax = 0.4 pmol/mg protein) was inhibited by picrotoxin and related convulsants and by barbiturates and related depressants that interact with gamma-aminobutyric acid and benzodiazepine receptors via the picrotoxinin binding site. The convulsant/barbiturate binding activity chromatographed on gel filtration as a single peak coinciding with the benzodiazepine/gamma-aminobutyric acid receptor protein complex.     

Recombinant extracellular domain of the three major subunits of GABAA receptor show comparable secondary structure and benzodiazepine binding properties

The three most widely expressed subunits of the GABAA receptor are alpha(1), beta(2), and gamma(2) subunits, and the major isoform in the human brain is a pentameric receptor composed of 2alpha(1)2beta(2)1gamma(2). Previously, we overexpressed the extracellular domain Q28-R248 of GABAA receptor alpha(1) subunit. In the present study, the homologous extracellular domains Q25-G243 of GABAA receptor beta(2) subunit and Q40-G273 of gamma(2) subunit were also obtained through overexpression in Escherichia coli. Successful production of recombinant beta(2) and gamma(2) subunit receptor protein domains facilitates the comparison of structural and functional properties of the three subunits. To this end, the secondary structures of the three fragments were measured using CD spectroscopy and the beta-strand contents calculated to be >30%, indicating a beta-rich structure for all three fragments. In addition, the benzodiazepine (BZ)-binding affinity of the recombinant fragments were measured using fluorescence polarization to be 2.16 microM, 3.63 microM, and 1.34 microM for the alpha(1), beta(2), and gamma(2) subunit fragments, respectively, indicating that all three homomeric assemblies, including that of the beta(2) subunit, generally not associated with BZ binding, can bind BZ in the micromolar range. The finding that the BZ binding affinity of these recombinant domains was highest for the gamma(2) subunit and lowest for the beta(2) subunit is consistent with results from previous binding studies using hetero-oligomeric receptors. The present results exemplify the effective approach to characterize and compare the three major subunits of the GABAA receptor, for two of which the overexpression in E. coli is reported for the first time.     

Multiple receptor states are required to describe both kinetic binding and activation of neutrophils via N-formyl peptide receptor ligands

It is well-established that the binding of N-formyl peptides to the N-formyl peptide receptor on neutrophils can be described by a kinetic scheme that involves two ligand-bound receptor states, both a low affinity ligand-receptor complex and a high affinity ligand-receptor complex, and that the rate constants describing ligand-receptor binding and receptor affinity state interconversion are ligand-specific. Here we examine whether differences due to these rate constants, i.e. differences in the numbers and lifetimes of particular receptor states, are correlated with neutrophil responses, namely actin polymerization and oxidant production. We find that an additional receptor state, one not discerned from kinetic binding assays, is required to account for these responses. This receptor state is interpreted as the number of low affinity bound receptors that are capable of activating G proteins; in other words, the accumulation of these active receptors correlates with the extent of both responses. Furthermore, this analysis allows for the quantification of a parameter that measures the relative strength of a ligand to bias the receptor into the active conformation. A model with this additional receptor state is sufficient to describe response data when two ligands (agonist/agonist or agonist/antagonist pairs) are added simultaneously, suggesting that cells respond to the accumulation of active receptors regardless of the identity of the ligand(s).     

A role for a helical connector between two receptor binding sites of a long-chain peptide hormone

The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N- and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible epsilon-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC(50): 100-300 nM) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC(50): 0.6-50 nM), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency.     

Identification of binding sites for both dsRBMs of PKR on kinase-activating and kinase-inhibiting RNA ligands

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR has a double-stranded RNA-binding domain (dsRBD) composed of two copies of the dsRNA binding motif (dsRBM). PKR's dsRBD is involved in the regulation of the enzyme as dsRNAs of cellular and viral origins bind to the dsRBD, leading to either activation or inhibition of PKR's kinase activity. In this study, we site-specifically modified each of the dsRBMs of PKR's dsRBD with the hydroxyl radical generator EDTA small middle dotFe and performed cleavage studies on kinase-activating and kinase-inhibiting RNAs. These experiments led to the identification of binding sites for the individual dsRBMs on various RNA ligands including a viral activating RNA (TAR from HIV-1), a viral inhibiting RNA (VA(I) RNA from adenovirus), an aptamer RNA that activates PKR, and a small synthetic inhibiting RNA. These results indicate that some RNAs interact only with one dsRBM, while others can bind both dsRBMs of PKR. In addition, EDTA small middle dotFe modification coupled with site-directed mutagenesis was used to assess the extent of cooperativity in the binding of the two dsRBMs. These experiments support the hypothesis that simultaneous binding of both dsRBMs of PKR occurs on kinase activating RNA ligands.     

Automated identification of binding sites for phosphorylated ligands in protein structures

Phosphorylation is a crucial step in many cellular processes, ranging from metabolic reactions involved in energy transformation to signaling cascades. In many instances, protein domains specifically recognize the phosphogroup. Knowledge of the binding site provides insights into the interaction, and it can also be exploited for therapeutic purposes. Previous studies have shown that proteins interacting with phosphogroups are highly heterogeneous, and no single property can be used to reliably identify the binding site. Here we present an energy‐based computational procedure that exploits the protein three‐dimensional structure to identify binding sites involved in the recognition of phosphogroups. The procedure is validated on three datasets containing more than 200 proteins binding to ATP, phosphopeptides, and phosphosugars. A comparison against other three generic binding site identification approaches shows higher accuracy values for our method, with a correct identification rate in the 80–90% range for the top three predicted sites. Addition of conservation information further improves the performance. The method presented here can be used as a first step in functional annotation or to guide mutagenesis experiments and further studies such as molecular docking. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

A peptide core motif for binding to heterotrimeric G protein alpha subunits

Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to Galpha(i1). The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of Galpha(i1) and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with Galpha subunits representing all four G protein classes, acting as a core motif for Galpha interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (Galpha(i/0)-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to Galpha(i1-3) in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the Galpha subunits and excludes association with Gbetagamma. R6A-Galpha(i1) complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg(2+), suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using Galpha(i1)/Galpha(s) chimeras identify two regions of Galpha(i1) (residues 1-35 and 57-88) as determinants for strong R6A-G(ialpha1) interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states.     

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.     

Filter assay method for the estrogen receptor protein: Detection of both filled and unfilled estrogen binding sites

New filter assay methods are presented for quantitating both cytoplasmic and nuclear forms of the estrogen receptor protein. These methods exploit the strong adsorption of this protein to glass-fiber filters, which appears to occur without loss of steroid binding affinity. A “direct assay protocol” is described that detects only unfilled (nonliganded) estrogen binding sites. In addition, a convenient “exchange assay protocol” has been developed that detects, in addition, those receptors present whose binding sites have already bound nonradioactive estradiol. For the exchange assay, an extract containing receptor is adsorbed to a filter, which is washed free of unbound steroid and then equilibrated for a prolonged period with an excess volume of buffer containing radioactive estradiol. After brief washing in steroid-free buffer, the radioactivity adsorbed to the filter is measured to determine the amount of receptor present. These assays can be used at either 4 or 23°C, over a broad range of salt concentrations. The background of nonspecific binding is extremely low, due in part to the almost negligible affinity of free estradiol for the glass-fiber support.     

Identification of sequence segments forming the alpha-bungarotoxin binding sites on two nicotinic acetylcholine receptor alpha subunits from the avian brain

The relationship between neuronal alpha-bungarotoxin binding proteins (alpha BGTBPs) and nicotinic acetylcholine receptor function in the brain of higher vertebrates has remained controversial for over a decade. Recently, the cDNAs for two homologous putative ligand binding subunits, designated alpha BGTBP alpha 1 and alpha BGTBP alpha 2, have been isolated on the basis of their homology to the N terminus of an alpha BGTBP purified from chick brain. In the present study, a panel of overlapping synthetic peptides corresponding to the complete chick brain alpha BGTBP alpha 1 subunit and residues 166-215 of the alpha BGTBP alpha 2 subunits were tested for their ability to bind 125I-alpha BGT. The sequence segments corresponding to alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were found to consistently and specifically bind 125I-alpha BGT. The ability of these peptides to bind alpha BGT was significantly decreased by reduction and alkylation of the Cys residues at positions 190/191, whereas oxidation had little effect on alpha BGT binding activity. The relative affinities for alpha BGT of the peptide sequences alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were compared with those of peptides corresponding to the sequence segments Torpedo alpha 1-(181-200) and chick muscle alpha 1-(179-198). In competition assays, the IC50 for alpha BGTBP alpha 1-(181-200) was 20-fold higher than that obtained for the other peptides (approximately 2 versus 40 microM). These results indicate that alpha BGTBP alpha 1 and alpha BGTBP alpha 2 are ligand binding subunits able to bind alpha BGT at sites homologous with nAChR alpha subunits and that these subunits may confer differential ligand binding properties on the two alpha BGTBP subtypes of which they are components.     

Evidence for two progesterone receptor binding sites in murine mammary carcinomas

We have evaluated the progesterone receptor (PR) binding patterns in progestin-dependent and -independent murine mammary carcinomas; all variants regress completely after antiprogestin treatment. These studies revealed the presence of a high affinity, low capacity-binding site (K(d): 43 +/- 9 pM; Q=9 +/- 3 fmol/mg protein) and of the classical lower affinity, high capacity-binding site (K(d): 9.2 +/- 4.2 nM; Q=376 +/- 64 fmol/mg protein). These sites could also be detected in uterus. Antiprogestins were able to bind to both sites. In vitro, medroxyprogesterone acetate (MPA) was stimulatory along a biphasic curve with two slopes, one at very low concentrations (EC(50): 1.5 +/- 0.7 fM) and the other at values compatible with the described K(d) for the PR (EC(50): 0.33 +/- 0.3 nM).     

An integrated machine learning system to computationally screen protein databases for protein binding peptide ligands

A fairly large set of protein interactions is mediated by families of peptide binding domains, such as Src homology 2 (SH2), SH3, PDZ, major histocompatibility complex, etc. To identify their ligands by experimental screening is not only labor-intensive but almost futile in screening low abundance species due to the suppression by high abundance species. An ideal way of studying protein-protein interactions is to use high throughput computational approaches to screen protein sequence databases to direct the validating experiments toward the most promising peptides. Predictors with only good cross-validation were not good enough to screen protein databases. In the current study we built integrated machine learning systems using three novel coding methods and screened the Swiss-Prot and GenBank protein databases for potential ligands of 10 SH3 and three PDZ domains. A large fraction of predictions has already been experimentally confirmed by other independent research groups, indicating a satisfying generalization capability for future applications in identifying protein interactions.     

Streptolysin O: the C-terminal, tryptophan-rich domain carries functional sites for both membrane binding and self-interaction but not for stable oligomerization

Streptolysin O belongs to the class of thiol-activated toxins, which are single chain, four-domain proteins that bind to membranes containing cholesterol and then assemble to form large oligomeric pores. Membrane binding involves a conserved tryptophan-rich sequence motif located within the C-terminally located domain 4. In contrast, sites involved in oligomerization and pore formation have been assigned to domains 1 and 3, respectively. We here examined the functional properties of domain 4, which was recombinantly expressed with an N-terminal histidine tag for purification and an additional cysteine residue for covalent labeling. The fluorescently labeled fragment readily bound to membranes, but it did not form oligomers nor lyse cell membranes. Moreover, the labeled fragment did not detectably become incorporated into hybrid oligomers when combined with lytically active full-length toxin. However, when present in large excess over the active toxin, the domain 4 fragment effected reduction of hemolytic activity and of functional pore size, which indicates interference with oligomerization of the lytically active species. Our findings support the notion that domain 4 of the streptolysin O molecule may fold autonomously, is essential for membrane binding and is capable not of irreversible but of reversible association with the entire toxin molecule.     

Discovering amino acid patterns on binding sites in protein complexes

Discovering amino acid (AA) patterns on protein binding sites has recently become popular. We propose a method to discover the association relationship among AAs on binding sites. Such knowledge of binding sites is very helpful in predicting protein-protein interactions. In this paper, we focus on protein complexes which have protein-protein recognition. The association rule mining technique is used to discover geographically adjacent amino acids on a binding site of a protein complex. When mining, instead of treating all AAs of binding sites as a transaction, we geographically partition AAs of binding sites in a protein complex. AAs in a partition are treated as a transaction. For the partition process, AAs on a binding site are projected from three-dimensional to two-dimensional. And then, assisted with a circular grid, AAs on the binding site are placed into grid cells. A circular grid has ten rings: a central ring, the second ring with 6 sectors, the third ring with 12 sectors, and later rings are added to four sectors in order. As for the radius of each ring, we examined the complexes and found that 10Å is a suitable range, which can be set by the user. After placing these recognition complexes on the circular grid, we obtain mining records (i.e. transactions) from each sector. A sector is regarded as a record. Finally, we use the association rule to mine these records for frequent AA patterns. If the support of an AA pattern is larger than the predetermined minimum support (i.e. threshold), it is called a frequent pattern. With these discovered patterns, we offer the biologists a novel point of view, which will improve the prediction accuracy of protein-protein recognition. In our experiments, we produced the AA patterns by data mining. As a result, we found that arginine (arg) most frequently appears on the binding sites of two proteins in the recognition protein complexes, while cysteine (cys) appears the fewest. In addition, if we discriminate the shape of binding sites between concave and convex further, we discover that patterns {arg, glu, asp} and {arg, ser, asp} on the concave shape of binding sites in a protein more frequently (i.e. higher probability) make contact with {lys} or {arg} on the convex shape of binding sites in another protein. Thus, we can confidently achieve a rate of at least 78%. On the other hand {val, gly, lys} on the convex surface of binding sites in proteins is more frequently in contact with {asp} on the concave site of another protein, and the confidence achieved is over 81%. Applying data mining in biology can reveal more facts that may otherwise be ignored or not easily discovered by the naked eye. Furthermore, we can discover more relationships among AAs on binding sites by appropriately rotating these residues on binding sites from a three-dimension to two-dimension perspective. We designed a circular grid to deposit the data, which total to 463 records consisting of AAs. Then we used the association rules to mine these records for discovering relationships. The proposed method in this paper provides an insight into the characteristics of binding sites for recognition complexes.     

Structural elements of the gamma-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands

gamma-aminobutyric acid type A (GABAA) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the alpha5 subunit were substituted by their corresponding alpha1 residues. Given the high affinity and selectivity of alpha1-containing compared with alpha5-containing GABAA receptors for zolpidem, mutated alpha5 subunits were co-expressed with beta2 and gamma2 subunits, and the affinity of recombinant receptors for zolpidem was measured. One alpha5 mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the alpha1 subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, alpha5P162T and alpha5E200G, might alter binding pocket conformation, whereas alpha5T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem. These three amino acid substitutions also influenced receptor affinity for CL218872. Our data thus suggest that corresponding amino acids of the alpha1 subunit, particularly alpha1-Ser204, are the crucial residues influencing ligand selectivity at the binding pocket of alpha1-containing receptors, and a model of this binding pocket is presented.     

Graphical analysis of binding data reflecting competition between two ligands for the same acceptor sites

A theoretical expression is derived for the analysis of results from competitive binding studies in which two multivalent ligands compete for acceptor sites, and a linear transform is suggested for simple graphical representation and assessment of experimental results. The protocol is illustrated by application to competitive binding data, obtained by ultrafiltration, on the interactions of bovine serum albumin with two structurally similar organic anions, methyl orange and methyl red. In a second experimental study the present approach is then used to establish that lactate dehydrogenase and aldolase compete for the same myofibrillar sites of bovine cardiac muscle. Finally, numerically simulated behavior of systems with additional binding sites for either ligand is used to emphasize that the criterion for classical (complete) competition is agreement between an experimentally determined equilibrium constant for ligand binding and the apparent value deduced from competitive binding studies. Nevertheless, the present analysis of competitive binding data should still offer considerable scope for screening quantitatively the cross-reactivities of drug and antigen analogs for their respective specific protein-acceptor sites.     

Quantitative structure-activity relationship studies on benzodiazepine receptor binding: recognition of active sites in receptor and modelling of interaction.

A quantitative structure-activity relationship study was carried out for the binding of a series of 'classical' benzodiazepines (BZs) and some beta-carbolines with BZ receptors to investigate the active sites in the latter and the nature of the binding of compounds with them. Using the Hansch approach, an attempt was made to correlate binding affinities of compounds with various physico-chemical and electronic properties of substituents. The correlations obtained showed the main roles were played by the hydrophobic constant pi and the Hammett constant sigma (an electronic parameter) of various substituents. This led to the suggestion that BZ receptors have many additional hydrophobic, hydrogen bonding and polar sites other than those suggested by Hollinshead et al. (1990). From the present study, the Hollinshead model of interaction was found to be inadequate to account fully for the binding of all types of compounds.