Functional requirements for specific ligand recognition by a biotin-binding RNA pseudoknot

Ligand-binding RNAs and DNAs (aptamers) isolated by in vitro selection from random sequence pools provide convenient model systems for understanding the basic relationships between RNA structure and function. We describe a series of experiments that define the functional requirements for an RNA motif that specifies high-affinity binding to the carboxylation cofactor biotin. A simple pseudoknot containing an adenosine-rich loop accounts for binding in all independently derived aptamers selected to bind biotin, suggesting that it alone represents a global optimum for recognition of this particular nonaromatic, electrostatically neutral ligand. In contrast to virtually all previously identified aptamers, unpaired nucleotides make up a small fraction of the binding motif. Instead, the identity of 14 nucleotides involved in base pairing is highly conserved among functional clones and their substitution by nonidentical base pairs significantly reduces or eliminates binding. Chemical probing is consistent with the predicted pseudoknot motif and indicates that relatively little change in structure accompanies ligand binding, a strong contrast with results for other aptamers. Competition experiments suggest that the aptamer recognizes all parts of the biotin ligand, including its thiophane ring and fatty acid tail. Two alternative modes of binding are suggested by a three-dimensional model of the pseudoknot, both of which entail significant interactions with base-paired nucleotides.     

Interaction of biotin with streptavidin. Thermostability and conformational changes upon binding

The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature Tm, of thermally induced denaturation of STV from 75 degrees C in unliganded protein to 112 degrees C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a Ka approximately 10(15) reproduce the behavior observed, but the estimated values of Tm are 15-20 degrees C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.     

Patterning of immobilized antibody layers via photolithography and oxygen plasma exposure

A novel technique for patterning immobilized antibody layers based upon photolithography and oxygen plasma exposure has been developed. Mouse monoclonal antibodies specific for thiabendazole (a post-harvest fungicide and veterinary anthelmintic) were covalently linked through free amine groups to aminosilanized silicon dioxide films using glutaraldehyde. Immobilized antibody layers were stabilized with sucrose, dehydrated, and stored refrigerated with desiccant. Photolithographic patterning was performed with a positive photoresist with modified bake temperatures and times, selective UV exposure with a contact mask, and aqueous alkaline solubilization of exposed resist. Exposed regions of immobilized antibody were then removed by exposure to a low power, radio frequency oxygen discharge. Residual resist was stripped with acetone. Successful patterning was demonstrated by challenging surfaces with goat anti-mouse antibody conjugated to tetramethylrhodamine isothiocyanate. Sucrose stabilization was necessary for antibody to undergo photoresist processing without loss of binding activity. Challenge with enzyme linked antigen of oxygen plasma exposed antibody layers demonstrated that plasma treatment completely neutralized antibody capture ability. Ellipsometry measurements of oxygen plasma exposed antibody layers indicated complete removal of immobilized antibodies. Fluorescent imaging demonstrated smallest line widths of 2-3 microns.     

Identification of amino acids in the N-terminal domain of corticotropin-releasing factor receptor 1 that are important determinants of high-affinity ligand binding

The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists: one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76-84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg76 or Asn81 but not Gly83 of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100-1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding.     

Unwinding of origin-specific structures by human replication protein A occurs in a two-step process

The simian virus 40 (SV40) large tumor antigen(T antigen) has been shown to induce the melting of 8 bp within the SV40 origin of replication. We found previously that a 'pseudo-origin' DNA molecule (PO-8) containing a central 8 nt single-stranded DNA (ssDNA) bubble was efficiently bound and denatured by human replication protein A (hRPA). To understand the mechanism by which hRPA denatures these pseudo-origin molecules, as well as the role that hRPA plays during the initiation of SV40 DNA replication, we characterized the key parameters for the pseudo-origin binding and denaturation reactions. The dissociation constant of hRPA binding to PO-8 was observed to be 7.7 x 10(-7) M, compared to 9.0 x 10(-8) M for binding to an identical length ssDNA under the same reaction conditions. The binding and denaturation of PO-8 occurred with different kinetics with the rate of binding determined to be approximately 4-fold greater than the rate of denaturation. Although hRPA binding to PO-8 was relatively temperature independent, an increase in incubation temperature from 4 to 37 degreesC stimulated denaturation nearly 4-fold. At 37 degreesC, denaturation occurred on approximately 1/3 of those substrate molecules bound by hRPA, showing that hRPA can bind the pseudo-origin substrate without causing its complete denaturation. Tests of other single-stranded DNA-binding proteins (SSBs) over a range of SSB concentrations revealed that the ability of the SSBs to bind the pseudo-origin substrate, rather than denature the substrate, correlated best with the known ability of these SSBs to support the T antigen-dependent SV40 origin-unwinding activity. Our data indicate that hRPA first binds the DNA substrate using a combination of contacts with the ssDNA bubble and duplex DNA flanks and then, on only a fraction of the bound substrate molecules, denatures the DNA substrate.     

Functional analysis of GnRH receptor ligand binding using biotinylated GnRH derivatives

The objective of this study was to determine whether the gonadotrophin-releasing hormone (GnRH) ligand binds to the GnRH receptor (GnRH-R) with either the N- and C-termini or the beta-II turn pointing towards the cell. The functionality of GnRH and two biotinylated GnRH derivatives, biotin [D-Lys6]GnRH and biotin [Gln1]GnRH biotinylated at positions 6 and 1, respectively was assessed. Streptavidin was also used in combination with these peptides to investigate the effects of the steric hindrance caused by this molecule on ligand binding when bound to the biotin molecules at the two positions. GnRH bound to the receptor with high affinity, which was not affected by the addition of streptavidin. Both the biotinylated derivatives bound to the receptor though with lower affinities than GnRH. The biotin [D-Lys6]GnRH-streptavidin complex bound to the receptor albeit with lower affinity compared to biotin [D-Lys6]GnRH only, although it maintained its ability to cause receptor internalisation. The ability of the biotin [Gln1]GnRH to bind to the receptor was abolished in the presence of excess streptavidin. Both GnRH and biotin [D-Lys6]GnRH stimulated total inositol phosphate production whereas biotin [Gln1]GnRH exhibited GnRH antagonist activity. It appears that the small biotin molecule can be accommodated within the binding pore when attached to position 1 of the ligand but not when complexed to streptavidin. The fact that biotin [D-Lys6]GnRH maintains functionality when complexed to streptavidin while biotin [Gln1]GnRH does not, suggests that the N- and possibly the C-termini are required for receptor binding. Thus the most likely binding orientation for the ligand is with the N- and C-termini pointing inwards with the residue at position 6 pointing away from the binding site.     

A streptavidin mutant with altered ligand-binding specificity

The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between biotin and these biotin analogs. Free energy calculations predicted that the introduction of a negative charge at the position of S27 plus the mutation N23A should disrupt two of the three hydrogen bonds between natural streptavidin and the ureido oxygen of biotin. In contrast, the imino hydrogen of 2-iminobiotin should form a hydrogen bond with the side chain of an acidic amino acid at position 27. This should reduce the biotin-binding affinity by approximately eight orders of magnitude, while leaving the affinities for these biotin analogs virtually unaffected. In good agreement with these predictions, a streptavidin mutant with the N23A and S27D substitutions binds 2-iminobiotin with an affinity (Ka) of 1 x 10(6) M-1, two orders of magnitude higher than that for biotin (1 x 10(4) M-1). In contrast, the binding affinity of this streptavidin mutant for diaminobiotin (2.7 x 10(4) M-1) was lower than predicted (2.9 x 10(5) M-1), suggesting the position of the diaminobiotin in the biotin-binding site was not accurately determined by modeling.     

The use of high-flow high performance liquid chromatography coupled with positive and negative ion electrospray tandem mass spectrometry for quantitative bioanalysis via direct injection of the plasma/serum samples

Two bioanalytical methods have been developed and validated utilizing high flow high performance liquid chromatography (HPLC) for on-line purification of plasma and serum samples and electrospray tandem mass spectrometry for detection and quantitation. Each plasma or serum sample, after mixing with an aqueous solution of the internal standard, was injected into a small diameter (1 x 50 mm) column packed with large particles of OASIS (30 microns), with a 100% aqueous mobile phase at a high flow rate (3-4 mL/min). The combination of the high linear speed (6-8 cm/s) of the aqueous mobile phase and the large particle size resulted in the rapid passage of the proteins and other large biomolecules through the column while the small-molecule analytes were retained on the column. During this purification period, the HPLC effluent was directed to waste. After the purification step, the HPLC mobile phase was rapidly changed from 100% aqueous to < or = 100% organic, the flow was reduced to 0.5-0.8 mL/min, and the column effluent was directed towards the mass spectrometer. The small molecule analytes were eluted during this period. In the method developed and validated for the quantitative determination of compound I in rat plasma (method A), the same OASIS column (1 x 50 mm, 30 microns) served as the purification and analytical (elution) column. In the method developed for the simultaneous determination of pravastatin and its positional isomer biotransformation product (SQ-31906) in human serum (method B), the purification column was connected to a conventional C18 analytical column (3.9 x 50 mm, 5 microns) to achieve the required chromatographic separation between the two isomers. For method A, where 50 microL of rat plasma mixed 1:1 with water containing the internal standard was injected, the standard curve range was 1 to 1,000 ng/mL. For method B, where 200 microL of a human serum sample mixed 4:1 with water containing the internal standard was injected, the standard curve range was 0.5 to 100 ng/mL. The total analysis time for each method was < or = 5 min per sample. The accuracy, inter-day precision and intra-day precision were within 10% for both methods.     

Oligonucleotide inhibitors of human thrombin that bind distinct epitopes

Thrombin, a multifunctional serine protease, recognizes multiple macromolecular substrates and plays a key role in both procoagulant and anticoagulant functions. The substrate specificity of thrombin involves two electropositive surfaces, the fibrinogen-recognition and heparin-binding exosites. The SELEX process is a powerful combinatorial methodology for identifying high-affinity oligonucleotide ligands to any desired target. The SELEX process has been used to isolate single-stranded DNA ligands to human thrombin. Here, a 29-nucleotide single-stranded DNA ligand to human thrombin, designated 60-18[29], with a Kd of approximately 0.5 nM is described. DNA 60-18[29] inhibits thrombin-catalyzed fibrin clot formation in vitro. Previously described DNA ligands bind the fibrinogen-recognition exosite, while competition and photocrosslinking experiments indicate that the DNA ligand 60-18[29] binds the heparin-binding exosite. DNA 60-18[29] is a quadruplex/duplex with a 15-nucleotide "core" sequence that has striking similarity to previously described DNA ligands to thrombin, but binds with 20 to 50-fold higher affinity. The 15-nucleotide core sequence has eight highly conserved guanine residues and forms a G-quadruplex structure. A single nucleotide within the G-quadruplex structure can direct the DNA to a distinct epitope. Additional sequence information in the duplex regions of ligand 60-18[29] contribute to greater stability and affinity of binding to thrombin. A low-resolution model for the interaction of DNA 60-18[29] to human thrombin has been proposed.     

Mutation of lysines in a plasminogen binding region of streptokinase identifies residues important for generating a functional activator complex

Through a unique but poorly understood mechanism, streptokinase (SK) interacts with human plasminogen to generate an "activator complex" that efficiently cleaves substrate plasminogen molecules. Previous studies have suggested that lysine residues in SK may play a role in the binding and function of the activator complex. To investigate this hypothesis, 10 different lysine residues in the plasminogen binding region of SK were altered to construct 8 recombinant (r) SK mutants. Only one double mutant, rSKK256,257A (replacing Lys with Ala at residues 256 and 257), showed a statistically significant reduction (63%) in binding affinity for Glu-plasminogen. This mutant also displayed a lagtime in the appearance of maximal activity, and modest impairments (2-5-fold) in kinetic parameters for amidolytic and plasminogen activator activity compared to rSK. In contrast, another mutant, rSKK332,334A, formed an activator complex with profound and nearly selective defects in the catalytic processing of substrate plasminogen molecules. When compared to rSK in kinetic assays of plasminogen activation, the rSKK332,334A mutant formed an activator complex that bound substrate plasminogens normally (normal K(m), but its ability to activate or cleave these molecules (kcat) was reduced by 34-fold. In contrast, in amidolytic assays, the kinetic parameters of rSKK332,334A showed only minor differences (< 2-fold) from rSK. Similarly, the binding affinity of this mutant to human Glu-plasminogen was indistinguishable from rSK [(2.6 +/- 0.8) x 10(9) vs (2.4 +/- 0.2) x 10(9) M-1, respectively]. In summary, these experiments have identified lysine residues in a plasminogen binding region of SK which appear to be necessary for normal high-affinity binding to plasminogen, and for the efficient catalytic processing of substrate plasminogen molecules by the activator complex.     

Morphological changes induced by colchicine in the chick optic cup in early stages of development. A stereological study

In this study chick embryo optic cups at HH stage 13 of development were analyzed under normal conditions and after inoculation with colchicine for 1, 2, 4, and 8 h. Several changes were seen after these periods of treatment: 1) modifications of the structure, with thicker regions in the cup and a general decrease in the total volume according to the duration of exposure to the drug (about 4 times less than normal, 5,035 x 10(3) microns 3 vs 1,334 x 10(3) microns 3 after 8 h of treatment); 2) enlargement of the ventricular cavity and its closure, due to failure of approximation of retinal and pigmentary layers; 3) failure of lens development, with delay and impairment of pit formation and deformation of all structures; lens volume was less than normal (about 4 times less, 2,148 x 10(3) microns 3 vs 658 x 10(3) microns 3 after 8 h of treatment); 4) a general segregation of the cells making up the structure, principally in the more active proliferating zones. The local alterations found are described.     

Fabrication and selective surface modification of 3-dimensionally textured biomedical polymers from etched silicon substrates

A new method is described for producing biomedically relevant polymers with precisely defined micron scale surface texture in the x, y, and z planes. Patterned Si templates were fabricated using photolithography to create a relief pattern in photoresist with lateral dimensions as small as 1 micron. Electroless Ni was selectively deposited in the trenches of the patterned substrate. The Ni served as a resilient mask for transferring the patterns onto the Si substrate to depths of up to 8.5 microns by anisotropic reactive ion etching with a fluorine-based plasma. The 3-dimensional (3-D) textured silicon substrates were used as robust, reusable molds for pattern transfer onto poly (dimethyl siloxane), low density poly (ethylene), poly (L-lactide), and poly (glycolide) by either casting or injection molding. The fidelity of the pattern transfer from the silicon substrates to the polymers was 90 to 95% in all three planes for all polymers for more than 60 transfers from a single wafer, as determined by scanning electron microscopy and atomic force microscopy. Further, the 3-D textured polymers were selectively modified to coat proteins either in the trenches or on the mesas by capillary modification or selective coating techniques. These selectively patterned 3-D polymer substrates may be useful for a variety of biomaterial applications.     

Effectiveness of influenza vaccine in reducing hospital admissions in people with diabetes

By radioligand binding followed by Scatchard analysis, we characterized and quantitated the specific binding sites for bFGF on cultured trabecular meshwork cells obtained from freshly enucleated porcine eyes. We detected two binding sites: 1.67 x 10(4) +/- 5.75 x 10(2) high-affinity receptors per cell with a Kd of 33.4 +/- 7.90 pM, and 1.70 x 10(4) +/- 7.57 x 10(5) low-affinity binding sites per cell with a Kd of 3.84 +/- 1.41 nM. At low concentrations of 125I-bFGF (< 1.50 ng ml-1), binding was primarily determined by the high-affinity receptors and, at high concentrations (> 2.50 ng ml-1), binding was dependent on the low-affinity binding sites. By phase-contrast time-lapse video micrography and sequential photomicrography, we demonstrated that at a concentration of 1 ng ml-1, bFGF significantly stimulated the rate of mitosis of the trabecular meshwork cells in G0-phase compared with control cultures maintained in serum-free medium alone. Treatment with higher concentrations of bFGF did not reveal more potent effects on these cells. Our findings demonstrate that trabecular meshwork cells do possess low- and high-affinity receptors for bFGF and that bFGF induces these cells in vitro to re-enter the cell cycle. Because the low-affinity interactions of 125I-bFGF were reduced by 75% following pretreatment of the trabecular meshwork cells with heparinase, these sites represent cell-associated heparin-like molecules and heparan sulfate proteoglycans, and may control the bioavailability of bFGF to ocular tissues. Heparinase treatment also resulted in a 30% reduction in high-affinity binding, which may be secondary to the decreased low-affinity binding. This finding agrees with the well-established scheme for bFGF-receptor interaction. We conclude that bFGF at the concentration present in aqueous humor is capable of stimulating the mitotic activity of trabecular meshwork cells in vitro, suggesting a possible paracrine role of aqueous humour bFGF in vivo. The results obtained in this study, together with our previous findings on bFGF mRNA expression by trabecular meshwork cells and protein deposition in this tissue, also indicates that trabecular cells of the eye may utilize bFGF by an autocrine mechanism.     

Nonequilibrium analysis alters the mechanistic interpretation of inhibition of acetylcholinesterase by peripheral site ligands

The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge with a catalytic triad characteristic of serine hydrolases, and a peripheral site at the mouth of the gorge some 10-20 A from the acylation site. Many ligands that bind exclusively to the peripheral site inhibit substrate hydrolysis at the acylation site, but the mechanistic interpretation of this inhibition has been unclear. Previous interpretations have been based on analyses of inhibition patterns obtained from steady-state kinetic models that assume equilibrium ligand binding. These analyses indicate that inhibitors bound to the peripheral site decrease acylation and deacylation rate constants and/or decrease substrate affinity at the acylation site by factors of up to 100. Conformational interactions have been proposed to account for such large inhibitory effects transmitted over the distance between the two sites, but site-specific mutagenesis has failed to reveal residues that mediate the proposed conformational linkage. Since examination of individual rate constants in the AChE catalytic pathway reveals that assumptions of equilibrium ligand binding cannot be justified, we introduce here an alternative nonequilibrium analysis of the steady-state inhibition patterns. This analysis incorporates a steric blockade hypothesis which assumes that the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Simulations based on this nonequilibrium steric blockade model were in good agreement with experimental data for inhibition by the peripheral site ligands propidium and gallamine at low concentrations of either acetylthiocholine or phenyl acetate if binding of these ligands slows substrate association and dissociation rate constants by factors of 5-70. Direct measurements with the acylation site ligands huperzine A and m-(N,N, N-trimethylammonio)trifluoroacetophenone showed that bound propidium decreased the association rate constants 49- and 380-fold and the dissociation rate constants 10- and 60-fold, respectively, relative to the rate constants for these acylation site ligands with free AChE, in reasonable agreement with the nonequilibrium steric blockade model. We conclude that this model can account for the inhibition of AChE by small peripheral site ligands such as propidium without invoking any conformational interaction between the peripheral and acylation sites.     

Steroid hormone receptor studies in melanoma model systems

The transplantable B-16 melanotic melanoma carried in syngeneic C57Bl/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrose-density gradient centrifugation of cytosol after incubation with [3H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5-35 fmoles per mg cytosol protein. Scatchard analysis of [3H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 x 10(-10) M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [3H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [3H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high-affinity sites with a dissociation constant of 2 x 10(-9) M. Binding levels from 70-610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.     

Novel synthetic inhibitors of selectin-mediated cell adhesion: synthesis of 1,6-bis[3-(3-carboxymethylphenyl)-4-(2-alpha-D- mannopyranosyloxy)phenyl]hexane (TBC1269)

Reports of a high-affinity ligand for E-selectin, sialyl di-Lewis(x) (sLe(x)Le(x), 1), motivated us to incorporate modifications to previously reported biphenyl-based inhibitors that would provide additional interactions with the protein. These compounds were assayed for the ability to inhibit the binding of sialyl Lewis(x) (sLe(x), 2) bearing HL-60 cells to E-, P-, and L-selectin fusion proteins. We report that dimeric or trimeric compounds containing multiple components of simple nonoligosaccharide selectin antagonists inhibit sLe(x)-dependent binding with significantly enhanced potency over the monomeric compound. The enhanced potency is consistent with additional binding interactions within a single selectin lectin domain; however, multivalent interaction with multiple lectin domains as a possible alternative cannot be ruled out. Compound 15e (TBC1269) showed optimal in vitro activity from this class of antagonists and is currently under development for use in the treatment of asthma.     

Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex

The high-affinity streptavidin-biotin complex is characterized by an extensive hydrogen-bonding network. A study of hydrogen-bonding energetics at the ureido oxygen of biotin has been conducted with site-directed mutations at Asn 23, Ser 27, and Tyr 43. A new competitive biotin binding assay was developed to provide direct equilibrium measurements of the alterations in Kd. S27A, Y43F, Y43A, N23A, and N23E mutants display DeltaDeltaG degrees at 37 degrees C relative to wild-type streptavidin of 2.9, 1.2, 2.6, 3.5, and 2.6 kcal/mol, respectively. The equilibrium-binding enthalpies for all of the mutants were measured by isothermal titration calorimetry, and the Y43A and N23A mutants display large decreases in the equilibrium binding enthalpy at 25 degrees C of 8.9 and 6.9 kcal/mol, respectively. The S27A and N23E mutants displayed small decreases in binding enthalpy of 1.6 and 0.9 kcal/mol relative to wild-type, while the Y43F mutant displayed a -2.6 kcal/mol increase in the binding enthalpy at 25 degrees C. At 37 degrees C, the Y43A and N23A mutants display decreases of 7.8 and 7.9 kcal/mol, respectively, while the S27A, N23E, and Y43F mutants displayed decreases of 4.9, 3.7, and 1.2 kcal/mol relative to wild-type. Kinetic analyses were also conducted to probe the contributions of the hydrogen bonds to the activation barrier. Wild-type streptavidin at 37 degrees C displays a koff of (4.1 +/- 0.3) x 10(-5) s-1, and the conservative Y43F, S27A, and N23A mutants displayed increases in koff to (20 +/- 1) x 10(-5) s-1, (660 +/- 40) x 10(-5) s-1, and (1030 +/- 220) x 10(-)5 s-1, respectively. The Y43A and N23E mutants displayed 93-fold and 188-fold increases in koff, respectively. Activation energies and enthalpies for each of the mutants were determined by transition-state analysis of the dissociation rate temperature dependence. All of the mutants except Y43F display large reductions in the activation enthalpy. The Y43F mutant has a more positive activation enthalpy, and thus a more favorable activation entropy that underlies the overall reduction in the activation barrier. For the most conservative mutant at each ureido oxygen hydrogen-bonding position, bound-state alterations account for most of the energetic changes in a single transition-state model, suggesting that the ureido oxygen hydrogen-bonding interactions are broken in the dissociation transition state.     

In vitro biotinylation provides quantitative recovery of highly purified active lactose permease in a single step

Consler et al. [Consler, T. G., Persson, B. L., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6934-6938] described a one-step purification of lactose permease, a hydrophobic membrane transport protein, from Escherichia coli. Permease constructs containing a biotin acceptor domain are biotinylated in vivo, followed by solubilization and avidin affinity purification. Although a high degree of purity is obtained, only about 15-20% of the permease is recovered due to incomplete biotinylation. In this communication, a simple modification is described that allows quantitative recovery of highly purified permease. Membranes containing permease with the biotin acceptor domain from the Klebsiella pneumoniae oxaloacetate decarboxylase are extracted with 5 M urea or treated with dicyclohexylcarbodiimide to inactivate F1/Fo ATPase and biotinylated in vitro with biotin ligase, ATP and d-biotin. Subsequently, the membranes are harvested, washed to remove free biotin and solubilized with 2% n-dodecyl-beta-D-maltopyranoside. Biotinylated permease is then purified in one step by affinity chromatography on monomeric avidin-Sepharose. The purified material is homogeneous and exhibits full activity with respect to ligand binding and counterflow.     

Towards causal inference in occupational cancer epidemiology--II. Getting the count right

Forty-five mole-rats, representing 4 chromosomal species (2n = 52, 54, 58, 60) of the superspecies Spalax ehrenbergi, were collected from 12 localities in Israel in 4 distinct climatic regions. Feces were examined for coccidian oocysts and 41 (91%) were infected; 26 (63%) had multiple infections of up to 5 coccidian species, 4 of which are described here as new species. Sporulated oocysts of Eimeria anzanensis n. sp. were ellipsoidal 18.3 x 12.5 microns (14-22 x 10-16) and had elongate-ovoidal sporocysts 7.3 x 4.9 microns (5-10 x 3-7); it occurred in 39 of 45 (87%) mole-rats, including all chromosomal species. Sporulated oocysts of Eimeria spalacensis n. sp. were ovoidal 23.4 x 18.3 microns (17-29 x 12-21) with ovoidal sporocysts 9.4 x 6.8 microns (6-12 x 4-10); it occurred in 7 of 45 (16%) mole-rats (2n = 54, 58, 60). Sporulated oocysts of Eimeria carmelensis n. sp. were subspheroidal to ellipsoidal 19.1 x 16.5 microns (14-25 x 11-20) and had sporocysts that were spheroidal to ellipsoidal 8.6 x 6.2 microns (6-13 x 4-8); it occurred in 5 of 45 (11%) mole-rats (2n = 58, 60). Sporulated oocysts of Isospora spalacensis n. sp. were ellipsoidal 14.6 x 11.0 microns (12-17 x 9-14) with ellipsoidal to ovoidal sporocysts 8.5 x 4.5 microns (7.5-11 x 4-7); it occurred in 5 of 45 (11%) mole-rats (2n = 58, 60). Twenty-five of 45 (56%) mole-rats (all 4 species) were infected with a previously described form, Eimeria elliptica Sayin, Dincer, and Meric, 1977.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular modeling of interleukin-8 receptor beta and analysis of the receptor-ligand interaction

A structural model of interleukin-8 receptor type beta (IL-8R-beta) was constructed based on the structure of bacteriorhodopsin. High temperature molecular dynamics simulations were performed to search the possible conformations of loop regions in IL-8R-beta which recognize the ligand. The crystal structure of interleukin 8 (IL-8) was used as a geometric constraint of the extracellular loop regions of IL-8R-beta in the conformational search. 500 complex structures were extracted from the dynamics trajectory and five plausible models were selected based on the binding energy and known experimental data. To study further the interaction between IL-8R-beta and its ligands, the complex of IL-8R-beta and platelet factor 4 (PF4) C-terminal peptide was also modeled by molecular dynamics simulations. From these models, the N-terminus, extracellular domain 3 and extracellular domain 4 of IL-8R-beta were found to be important for ligand binding. Key residues of these regions involved in ligand binding were characterized. These models provide insight into the structural basis of biological activity of IL-8 and PF4 and may guide the design of potential therapeutic agents targeting IL-8 receptors. Furthermore, the approach developed from this study may have implications for the understanding of other chemokine receptor-ligand interactions that have been recently suggested to be involved in HIV infection.