A two-site mechanism for ATP hydrolysis by the asymmetric Rep dimer P2S as revealed by site-specific inhibition with ADP-A1F4

The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reaction--the asymmetric Rep dimer state, P2S, where ss DNA [dT(pT)15] is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP-A1F4. We found that when ADP-A1F4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s(-1) per dimer (equivalent to wild-type P2S) was recovered. However, when the ADP-A1F4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP <==> E-ATP <==> E'-ATP <==> E'-ADP-Pi <==> E-ADP-Pi <==> E-ADP + Pi <==> E + ADP + Pi, with K1 = (2.0 +/- 0.85) x 10(5) M(-1), k2 = 22 +/- 3.5 s(-1), k(-2) < 0.12 s(-1), K3 = 4.0 +/- 0.4 (k3 > 200 s(-1)), k4 = 1.2 +/- 0.14 s(-1), k(-4) < 1.2 s(-1), K5 = 1.0 +/- 0.2 mM, and K6 = 80 +/- 8 microM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E'-ADP-Pi. In the context of our "subunit switching" model for Rep dimer translocation during processive DNA unwinding [Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol. 263, 411-422], this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.     

Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique

The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) containing all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate constants for association (k(a)) and dissociation (k(d)) of the duplex formation as well as the thermal stability (melting temperature, T(m)) of the duplexes have been determined. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences containing single mismatches at various positions in the center resulted in increased dissociation and decreased association rate constants. T(m) values for PNA x RNA duplexes are on average 4 degrees C higher than for PNA x DNA duplexes and follow quantitatively the same variation with mismatches as do the PNA x DNA duplexes. Also a faster k(a) and a slower k(d) are found for PNA x RNA duplexes compared to the PNA x DNA duplexes. An overall fair correlation between T(m), k(a), and k(d) is found for a series of PNA x DNA and PNA x RNA duplexes although the determination of k(a) seemed to be prone to artifacts of the method and was not considered capable of providing absolute values representing the association rate constant in bulk solution.     

Opposite base-dependent excision of 7,8-dihydro-8-oxoadenine by the Ogg1 protein of Saccharomyces cerevisiae

The yOgg1 protein of Saccharomyces cerevisiae is a DNA glycosylase/AP lyase that excises guanine lesions such as 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (me-Fapy-G) and incises apurinic/apyrimidinic sites (AP sites) in damaged DNA. The yOgg1 protein displays a marked preference for DNA duplexes containing 8-OxoG or AP sites placed opposite cytosine. In this paper, we show that yOgg1 can also excise an adenine lesion, 7,8-dihydro-8-oxoadenine (8-OxoA), when paired with cytosine or 5-methylcytosine. In contrast, yOgg1 does not release 8-OxoA when placed opposite thymine, adenine, guanine or uracil. The specificity constants (Kcat/Km) for repair of 8-OxoG/C and 8-OxoA/C duplexes are (50 +/- 18) x 10(-3) and (13 +/- 3) x 10(-3)/min/nM, respectively. The catalytic mechanism for strand cleavage at 8-OxoA/C involves excision of 8-OxoA by the DNA glycosylase activity of yOgg1, followed by incision at the newly formed AP site via a beta-elimination reaction. Furthermore, cleavage of 8-OxoA/C involves formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydride (NaBH4). The yOgg1 protein binds strongly to the 8-OxoA/C duplex, as demonstrated by an apparent dissociation constant (Kdapp) value of 45 nM, as determined by gel mobility shift assay. In contrast, the yOgg1 protein has a very low binding affinity for the 8-OxoA/T duplex, a Kdapp value of 680 nM, which in turn can explain the lack of repair of 8-OxoA in this duplex. The capacity of other DNA glycosylases/AP lyases to repair 8-OxoA has also been investigated. The results show that human hOgg1 protein efficiently repairs 8-OxoA placed opposite cytosine or 5-methylcytosine. On the other hand, the Fpg protein of Escherichia coli cleaves 8-OxoA/C at a very slow rate as compared with yOgg1.     

Equilibrium binding of estrogen receptor with DNA using fluorescence anisotropy

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (Kd) of 1.8 +/- 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the Kd for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.     

Kinetic mechanism for the sequential binding of two single-stranded oligodeoxynucleotides to the Escherichia coli Rep helicase dimer

Escherichia coli Rep helicase is a DNA motor protein that unwinds duplex DNA as a dimeric enzyme. Using fluorescence probes positioned asymmetrically within a series of single-stranded (ss) oligodeoxynucleotides, we show that ss-DNA binds with a defined polarity to Rep monomers and to individual subunits of the Rep dimer. Using fluorescence resonance energy transfer and stopped-flow techniques, we have examined the mechanism of ss-oligodeoxynucleotide binding to preformed Rep dimers in which one binding site is occupied by a single-stranded oligodeoxynucleotide, while the other site is free (P2S dimer). We show that ss-DNA binding to the P2S Rep dimer to form the doubly ligated P2S2 dimer occurs by a multistep process with the initial binding step occurring relatively rapidly with a bimolecular rate constant of k1 = approximately 2 x 10(6) M-1 s-1 [20 mM Tris (pH 7.5), 6 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4 degrees C]. A minimal kinetic mechanism is proposed which suggests that the two strands of ss-DNA bound to the Rep homodimer are kinetically distinct even within the P2S2 Rep dimer, indicating that this dimer is functionally asymmetric. The implications of these results for the mechanisms of DNA unwinding and translocation by the functional Rep dimer are discussed.     

Watson-Crick base-pairing properties of bicyclo-DNA

A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.     

Differential DNA recognition by the enantiomers of 1-Rh(MGP)2 phi: a combination of shape selection and direct readout

The enantiomers of the symmetric metallointercalator complex 1-Rh(MGP)2phi5+ [MGP = 4-(guanidylmethyl)-1,10-phenanthroline; phi = phenanthrenequinone diimine] bound to DNA decamer duplexes containing their respective 6 bp recognition sequences have been investigated using 1H NMR. Shape selection due to the chirality of the metal center and hydrogen-bonding contacts of ancillary guanidinium groups to 3'-G N7 atoms define the recognition by complexes which bind by intercalation to duplex DNA. The titration of Lambda-Rh into the self-complementary decamer containing the recognition sequence (5'-GACATATGTC-3', L1) resulted in one symmetric bound conformation observed in the 1H NMR spectrum, indicating that the DNA duplex retains its symmetry in the presence of the metal complex. Upfield chemical shifts of duplex imino protons and the disruption of the NOE base-sugar contacts defined the central T5-A6 intercalation site. The downfield shift of the G8 imino proton supports the conclusion that the pendant guanidinium arms make simultaneous H-bonding contacts to the N7 atoms of 3'-G8 bases on either side of the site. A variable-temperature study of a partially titrated sample (2:3 Lambda-Rh/L1) showed the exchange rate (kobs) at 298 K to be 68 s-1 and the activation barrier to exchange (DeltaG of association) to be 2.7 kcal/mol, a value comparable to the stacking energy of one base step. The results presented coupled with biochemical data are therefore consistent with binding models in which Lambda-1-Rh(MGP)2phi5+ (Lambda-Rh) traps the recognition site 5'-CATATG-3' in an unwound state, permitting intercalation centrally and hydrogen bonding to guanines at the first and sixth base pair positions. The data suggest a different model of binding and recognition by Delta-Rh. The titration of Delta-Rh into a DNA decamer containing the 6 bp recognition site (D1, 5'-CGCATCTGAC-3'; D2, 5'-GTCAGATGCG-3') resulted in two, distinct conformers, in slow exchange on the NMR time scale. The rate of exchange between the two conformers (kobs) at 298 K is 37 s-1, most likely due to partial dissociation between binding modes. The slower rate relative to Lambda-Rh association reflects the relative rigidity of the D1 and/or D2 sequence in comparison to L1. NOE cross-peaks between the intercalating phi ligand and protons of T5-C6, as well as the upfield shifts observed for imino protons at this step, serve to define the central T5-C6 step as the single site of intercalation. The downfield shift of the 3'-G imino protons indicates the complex makes hydrogen bond contacts with these bases. The complex, which is too small to span a 6 bp B-form DNA sequence, nonetheless makes major groove contacts with 3'-G bases to either side of the site. Notably, both 3'-guanine bases are necessary to impart site specificity and slow dissociation kinetics with the 5'-CATCTG-3' site, as evidenced by the extremely exchange-broadened two-dimensional NOESY spectra of Delta-Rh bound to modified duplexes containing N7-deazaguanine at either G8 or G18; the loss of one major groove contact completely abolishes specificity for 5'-CATCTG-3'. DNA chemical shifts upon binding and intermolecular NOE contacts therefore support a model in which Delta-Rh intercalates in one of two canted binding conformations. Within this model, each intercalation mode allows one guanidinium-guanine hydrogen bond at a time, while bringing the other arm close to the phosphate backbone.     

p53 DNA binding can be modulated by factors that alter the conformational equilibrium

The p53 tumor suppressor protein is a dimer of dimers that binds its consensus DNA sequence (containing two half-sites) as a pair of clamps. We show here that after one wild-type dimer of a tetramer binds to a half-site on the DNA, the other (unbound) dimer can be in either the wild-type or the mutant conformation. An equilibrium state between these two conformations exists and can be modulated by two types of regulators. One type modifies p53 biochemically and determines the intrinsic balance of the equilibrium. The other type of regulator binds directly to one or both dimers in a p53 tetramer, trapping each dimer in one or the other conformation. In the wild-type conformation, the second dimer can bind to the second DNA half-site, resulting in drastically enhanced stability of the p53-DNA complex. Importantly, a genotypically mutant p53 can also be in equilibrium with the wild-type conformation, and when trapped in this conformation can bind DNA.     

Detection of basepair substitution mutation at a frequency of 1 x 10(-7) by combining two genotypic selection methods, MutEx enrichment and allele-specific competitive blocker PCR

The detection of rare mutations has many important applications, including risk assessment of drugs and chemicals, measuring environmental exposures to genotoxins, and cancer cell detection. A sensitive genotypic selection method has been developed that combines two different mutant allele selection techniques, MutEx enrichment and allele-specific competitive blocker PCR (ACB-PCR). This method was developed and evaluated for the detection of a CAA --> AAA mutation at codon 61 of the mouse H-ras gene. The MutEx enrichment is based on MutS binding to a mismatched basepair in heteroduplex DNA. The bound MutS protects the mutant allele from degradation during subsequent exonuclease treatment. ACB-PCR preferentially amplifies a mutant allele in a PCR reaction using a primer that has more mismatches to the wild-type allele than the mutant allele. By combining these two approaches, the codon 61 mutation was detected at mutant fractions as low as 1 in 10(7). This sensitivity was achieved with the thermostable Thermus aquaticus MutS protein but not the Escherichia coli MutS protein. Using the combined approach, the average Pfu DNA polymerase error rate +/- the standard error of the mean for this particular basepair was estimated to be 8 +/- 3 x 10(-7) errors per duplication. The results indicate that MutEx/ACB-PCR is among the most sensitive genotypic selection methods for the detection of mutation.     

High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region

The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations. The relative dissociation constants for the mutant TAR RNAs were measured using a dual-label competition filter binding assay in which 35S-labelled wild-type TAR RNA (K1) was competed against 3H-labelled mutant TAR RNA (K2). The error in the self-competition experiment was usually less than 10% (e.g. K2/K1 = 1.07 +/- 0.05, n = 19) and the experimental data accurately matched theoretical curves calculated with fitted dissociation constants. Mutations in U23, a critical residue in the U-rich "bulge" sequence, or in either of the two base-pairs immediately above the "bulge", G26.C39 and A27.U38 reduced that affinity by 8- to 20-fold. Significant contributions to tat binding affinity were also made by the base-pairs located immediately below the bulge. For example, mutation of A22.U40 to U.A reduced tat affinity 5-fold, and mutation of G21.C41 to C.G reduced tat affinity 4-fold. The binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays. Each of the peptides showed significantly reduced affinities for wild-type TAR RNA compared to the tat protein. The ADP-2 (residues 43 to 72), ADP-3 (residues 48 to 72) and ADP-5 (residues 49 to 86) peptides were unable to discriminate between wild-type TAR RNA and TAR RNA mutants with the same fidelity as the tat protein. For example, these peptides showed no more than 3-fold reductions in affinity relative to wild-type TAR RNA for the U23-->C mutation in the bulge, or G26.G39-->C.G mutation in the stem of TAR RNA. By contrast, the ADP-I (residues 37 to 72), ADP-4 (residues 32 to 62) and ADP-6 (residues 32 to 72) peptides, which each carry amino acid residues from the "core" region of the tat protein have binding specificities that more closely resemble the protein. The ADP-4 and ADP-6 peptides showed between 4- and 7-fold reductions in affinity for the U23-->C or G26.C39-->C.G mutations. The ADP-1 peptide most closely resembles the protein in its binding specificity and showed 9-fold and 14-fold reductions in affinity for the two mutants, respectively. Chemical-modification interference assays using diethylpyrocarbonate (DEPC) and ethylnitrosourea (ENU) were also used to compare the binding properties of the tat protein and the tat-derived peptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Designed disulfide between N-terminal domains of lactose repressor disrupts allosteric linkage

Substitution of Cys for Val at position 52 of the lac repressor was designed to permit disulfide bond formation between the two N-terminal DNA binding domains that comprise an operator DNA binding site. This position marks the closest approach of these domains based on the x-ray crystallographic structures of the homologous purine holorepressor-operator complex and lac repressor-operator complex (Schumacher, M. A., Choi, K. Y., Zalkin, H., and Brennan, R. G. (1994) Science 266, 763-770; Lewis, M., Chang, G., Horton, N.C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Science 271, 1247-1254). The V52C mutation was generated by site-specific methods, and the mutant protein was purified and characterized. In the reduced form, V52C bound operator DNA with slightly increased affinity. Exposure to oxidizing conditions resulted in disulfide bond formation, and the oxidized protein bound operator DNA with approximately 6-fold higher affinity than wild-type protein. Inducer binding for both oxidized and reduced forms of V52C was comparable to wild-type lac repressor. In the presence of inducer, the reduced protein exhibited wild-type, diminished DNA binding. In contrast, DNA binding for the oxidized form was unaffected by inducer, even at 1 mM. Thus, the formation of the designed disulfide between Cys52 side chains within each dimer renders the protein-operator complex unresponsive to sugar binding, presumably by disrupting the allosteric linkage between operator and inducer binding.     

Clinical spectrum of nonmenstrual toxic shock syndrome (TSS): comparison with menstrual TSS by multivariate discriminant analyses

1. The present experiments were undertaken in order to characterize further the apparently irreversible inhibition of the contraction of depolarized rat aorta caused by lacidipine, a 1,4-dihydropyridine calcium antagonist. 2. We studied the effect of lacidipine on contraction evoked by 100 mM KCl solution in rat aorta, treated by N omega-nitro-L-arginine (0.1 mM), an inhibitor of nitric oxide (NO) synthesis. We compared the effect of prolonged depolarization on lacidipine and (+)-isradipine inhibition and the reversal of this inhibition after washout in the absence of dihydropyridines. Assuming that the onset of lacidipine-evoked inhibition was a pseudo-first order association kinetics, we estimated the dissociation rate constant (k-1 = 0.031 min-1), the association rate constant (k1 = 2.70 x 10(8) M-1 min-1) and the dissociation constant (KD = k-1/k1 = 115 pM) which was close to the IC50 value in steady-state conditions (160 pM). 3. The inhibitory effects of lacidipine and (+)-isradipine on rat aorta contraction were reversibly enhanced after preincubation with the drug in a 40 mM KCl-solution. Washout with drug-free 40 mM K(+)-depolarizing solution reversed inhibition in the (+)-isradipine-treated preparations, but not in the lacidipine-treated ones. 4. Radioligand binding studies were performed with [3H]-lacidipine and [3H]-isradipine in microsomes from rat aorta and rat ileum. Both ligands bound to a homogeneous population of binding sites (for[3H]-lacidipine: KD = 23 +/- 2.6 pM, Bmax = 380 +/- 21 fmol mg-1 protein in membranes from aorta; KD =23 +/- 3.1 pM, Bmax = 790 +/- 60 fmol mg-1 protein in membranes from ileum; for [3H]-isradipine:KD = 140 +/- 46 pM, Bmax = 350 +/- 64 fmol mg-1 protein in membrane from aorta; KD = 68 +/- 14 pM,Bmax = 760 +/- 75 fmol mg-1 protein in membranes from ileum). After isotopic dilution, [3H]-lacidipine and [3H]-isradipine dissociated according to a monoexponential kinetics. In membranes from ileum, the calculated dissociation rate constants (kappa_ 1) were 0.0257 min-1 and 0.0595 min-1, for [3H]-lacidipine and[3H]-isradipine, respectively.5. The non specific binding of [3H]-lacidipine and [3H]-isradipine, was measured in intact rat aorta preparations incubated under the conditions of the functional experiments, in the presence of nifedipine(1 microM). After incubation with [3H]-lacidipine 77.6 +/- 1.9 pM for 2 h the concentration of drug in the tissue was 15.15 +/- 1.18 fmol mg-1 w.wt. and still amounted to 7.24 +/- 0.61 fmol mg-1 w.wt. after 3.5 h washout in drug-free solution. After incubation with [3H]-isradipine 47.2 +/- 0.4 pM for 2 h it was 2.26 +/-0.07 fmol mg-1 w.wt. and was undetectable after 3.5 h washout in a drug-free solution.6. It is concluded that lacidipine interacts reversibly with dihydropyridine binding sites and that the apparent irreversible inhibition of contraction in depolarized preparations could be related to a nonspecific binding in a tissue compartment different from the plasma membrane.     

Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease

Phage T5 exonuclease is a 5'-->3'exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.     

Binding of guanine nucleotides to bronchial membranes: effect of maturation

Guanosine 5-[y-thio]triphosphate ([35S]GTP gamma S) binding to guinea pig bronchial membranes from immature and mature guinea pigs was rapid (Kon: 3.8 x 10(5) mol-1 min-1), saturable (Bmax: 160 pmoles/mg protein) and of high affinity (Kd: 0.6 microM). [35S]GTP gamma S rapidly dissociated in the absence of magnesium (Koff: 0.06 min-1), but 50 mM magnesium inhibited the dissociation. Maturation did not alter the affinity of the ligand, but Bmax (pmoles/mg DNA) was greater in preparations from mature animals (929 +/- 16 vs. 620 +/- 64). [35S]GTP gamma S was displaced by guanine nucleotides with a rank order of potency of GDP beta S = Gpp(NH)p > GDP > GTP, but not by ATP. We conclude that [35S]GTP gamma S is a specific and useful method to quantitate bronchial membrane-bound GTP-binding proteins. The technique shows that there is a significant increase in the cellular content of G-proteins during maturation.     

Renewed interest in granulocyte transfusion therapy

The kinetics of the interaction of heme with hemopexin and albumin was monitored by measuring the time dependence of changes in the Soret absorption spectra. Since the protein binding sites can only bind heme monomers, the binding kinetics apparently reflected the slow dissociation of heme dimers, resulting from dimer/monomer equilibria in aqueous heme solutions. The dissociation of heme dimers is characterized by the rate constant of (3-4) x 10(-3) s(-1). The measurements further revealed significant differences in the kinetic profiles (slowing down the binding interaction) that were dependent on the storage time of heme solutions at room temperature. These presumably responded to the gradual formation of higher aggregates of heme, which cannot dissociate into dimers/monomers. Alternatively, partial autooxidation of heme molecules could increase the stability of heme dimers and obstruct specific binding of heme to the proteins.     

Mutations in the carboxyl terminus of the agouti protein decrease agouti inhibition of ligand binding to the melanocortin receptors

Several mutations that cause ectopic expression of the agouti gene result in obesity, hyperinsulinemia, and yellow coat color. A candidate pathway for agouti induced obesity and hyperinsulinemia is through altered signaling by melanocortin receptors, as agouti normally regulates coat coloration through antagonism of melanocortin receptor 1. Furthermore, melanocortin peptides mediate functions including steroidogenesis, lipolysis, and thermoregulation. We report apparent inhibition dissociation constants for mouse and human agouti protein inhibition of ligand binding to the melanocortin receptors, to determine which of these receptors might be involved in agouti induced diabetes. The similarity in the apparent K(I) values for agouti inhibition of ligand binding to the brain melanocortin receptors 3 and 4 (mouse: K(I) app = 190 +/- 74 and 54 +/- 18 nM; human: K(I) app = 140 +/- 56 and 70 +/- 18 nM, respectively) suggests that the MC3-R is a potential candidate for a receptor mediating the effects of agouti protein overexpression. Agouti residues important for melanocortin receptor inhibition were identified through the analysis of deletion constructs and site-specific variants. Val83 is important for inhibition of binding to MC1-R (K(I) app for Val83Ala agouti increased 13-fold relative to wild-type protein). Arg85, Pro86, and Pro89 are important for selective inhibition of binding between MC1-R and MC3-R and MC4-R as their apparent K(I) values are essentially unchanged at MC1-R, while they have increased 6-10-fold relative to wild-type protein at MC3-R and MC4-R.     

Transferrins. Hen ovo-transferrin, interaction with bicarbonate and iron uptake

Fe(III) uptake by the iron-delivery and iron-scavenging protein, hen ovotransferrin has been investigated in vitro between pH 6.5 and 9. In the absence of any ferric chelate, apo-ovotransferrin loses two protons with K1a = 50 +/- 1 nM and K2a = 4.0 +/- 0.1 nM. These acid-base equilibria are independent of the interaction of the protein with bicarbonate. The interaction with bicarbonate occurs with two different affinity constants, KC = 9.95 +/- 0.15 mM and KN = 110 +/- 10 mM. FeNAc3 exchanges its Fe(III) with the C-site of the protein in interaction with bicarbonate, direct rate constants k1 = 650 +/- 25 M-1 s-1, reverse rate constant k-1 = (6.0 +/- 0.1) x 10(3) M-1 s-1 and equilibrium constant K1 = 0.11 +/- 0.01. This iron-protein intermediate loses then a single proton, K3a = 3.50 +/- 0.35 nM, and undergoes a first change in conformation followed by a two or three proton loss, first order rate constant k2 = 0.30 +/- 0.01 s-1. This induces a new modification in conformation followed by the loss of one or two protons, first order rate constant k3 = (1.50 +/- 0.05) x 10(-2) s-1. These modifications in the monoferric protein conformation are essential for iron uptake by the N-site of the protein. In the last step, the monoferric and diferric proteins attain their final state of equilibrium in about 15,000 s. The overall mechanism of iron uptake by ovotransferrin is similar but not identical to those of serum transferrin and lactoferrin. The rates involved are, however, closer to lactoferrin than serum transferrin, whereas the affinities for Fe(III) are lower than those of serum transferrin and lactoferrin. Does this imply that the metabolic function transferrins is more related to kinetics than to thermodynamics?     

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.