Characterization of deltoid, a chimeric protein containing the oligomerization site of hepatitis delta antigen

Both forms of the hepatitis delta antigen (HDAg) encoded by hepatitis delta virus are active only as oligomers. Previous studies showed that quadrin, a synthetic 50-residue peptide containing residues 12-60 from the N-terminus of HDAg, interferes with HDAg oligomerization, forms an alpha-helical coiled coil in solution, and forms a novel square octamer in the crystal consisting of four antiparallel coiled-coil dimers joined at the corners by hydrophobic binding of oligomerization sites located at each end of the dimers. We designed and synthesized deltoid (CH3CO-[Cys23]HDAg-(12-27)-seryl-tRNA synthetae-(59-65)-[Cys42]HDAg-(34-60)-Tyr-NH2), a chimeric protein that structurally resembles one end of the quadrin dimer and contains a single oligomerization site. The 51-residue chain of deltoid contains a seven-residue alpha-hairpin loop in place of the remainder of the quadrin dimer plus Cys12 and Cys31 for forming an intrachain disulfide bridge. Reduced, unbridged deltoid (Tm=61 degrees C, DeltaG(H2O)=-1.7 kcal mol(-1)) was less stable to denaturation by heat or guanidine HCl than oxidized, intrachain disulfide-bridged deltoid (Tm>80 degrees C, DeltaG(H2O)=-2.6 kcal mol(-1)). Each form is an alpha-helical dimer that reversibly dissociates into two monomers (Kd=80 microM).     

Effects of nucleotide- and aurodox-induced changes in elongation factor Tu conformation upon its interactions with aminoacyl transfer RNA. A fluorescence study

The effects of GDP and of aurodox (N-methylkirromycin) on the affinity of elongation factor Tu (EF-Tu) for aminoacyl-tRNA (aa-tRNA) have been quantified spectroscopically by using Phe-tRNA(Phe)-Fl8, a functionally active analogue of Phe-tRNA(Phe) with a fluorescein dye convalently attached to the s4U-8 base. The association of EF-Tu.GDP with Phe-tRNA(Phe)-Fl8 resulted in an average increase of 33% in fluorescein emission intensity. This spectral change was used to monitor the extent of ternary complex formation as a function of EF-Tu.GDP concentration, and hence to obtain a dissociation constant, directly and at equilibrium, for the EF-Tu.GDP-containing ternary complex. The Kd for the Phe-tRNA(Phe)-Fl8.EF-Tu.GDP complex was found to average 28.5 microM, more than 33,000-fold greater than the Kd of the Phe-tRNA(Phe)-Fl8.EF-Tu.GTP complex under the same conditions. In terms of free energy, the delta G degree for ternary complex formation at 6 degrees C was -11.5 kcal/mol with GTP and -5.8 kcal/mol with GDP. Thus, the hydrolysis of the ternary complex GTP results in a dramatic decrease in the affinity of EF-Tu for aa-tRNA, thereby facilitating the release of EF-Tu.GDP from the aa-tRNA on the ribosome. Aurodox (200 microM) decreased the Kd of the GDP complex by nearly 20-fold, to 1.46 microM, and increased the Kd of the GTP complex by at least 6-fold. The binding of aurodox to EF-Tu therefore both considerably strengthens EF-Tu.GDP affinity for aa-tRNA and also weakens EF-Tu.GTP affinity for aa-tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Thermodynamics of folding and binding in an affibody:affibody complex

Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K(d)=0.1 microM. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T(M) approximately 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with DeltaH degrees (bind) = -9.0 (+/-0.1) kcal mol(-1)(.) The heat capacity change DeltaC(P) degrees (,bind)=-0.43 (+/-0.01) kcal mol(-1) K(-1) is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-TDeltaS degrees (bind) = -0.6 (+/-0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.     

Suramin affects capsaicin responses and capsaicin-noxious heat interactions in rat dorsal root ganglia neurones

The effect of suramin, an inhibitor of G protein regulated signalling, was studied on the membrane currents induced by noxious heat and by capsaicin in cultured dorsal root ganglia neurones isolated from neonatal rats. Whole-cell responses induced by a heat ramp (24-52 degrees C) were little affected by suramin. The noxious heat-activated currents were synergistically facilitated in the presence of 0.3 microM capsaicin 13.2-fold and 6.3-fold at 40 degrees C and 50 degrees C, respectively. In 65% of neurones, the capsaicin-induced facilitation was inhibited by 10 microM suramin to 35 +/- 6% and 53 +/- 6% of control at 40 degrees C and 50 degrees C (S.E.M., n = 15). Suramin 30 microM caused a significant increase in the membrane current produced by a nearly maximal dose (1 microM) of capsaicin over the whole recorded temperature range (2.4-fold at 25 degrees C and 1.2-fold at 48 degrees C). The results demonstrate that suramin differentially affects the interaction between capsaicin and noxious heat in DRG neurones and thus suggest that distinct transduction pathways may participate in vanilloid receptor activation mechanisms.     

Functional interactions between smooth muscle myosin light chain kinase and calmodulin

Calmodulin (CaM) binding by turkey gizzard myosin light chain kinase (MLCK) causes subtle changes in the fluorescence emission and polarization excitation spectra of the enzyme. Fluorescence experiments using 9-anthroyl-choline (9AC), which competes with ATP in binding, demonstrate mutually stabilizing interactions between the CaM and ATP binding sites corresponding to delta G = -0.6 to -0.7 kcal/mol. Fluorescence titrations in the presence of 9AC or 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] confirm the stoichiometry of 1 mol of CaM/MLCK. Phosphorylation of MLCK has no effect on either the protein fluorescence or the binding of ATP and 9AC. The dissociation constant for the MLCL-CaM complex is increased approximately 500-fold on phosphorylation. Values of Kd for the phosphorylated enzyme range from 0.5 to 1.1 microM in 0.2 N KCl, pH 7.3, 25 degrees C. We showed competition between MLCK and other CaM binding proteins and peptides by using both fluorescence and catalytic activity measurements. Competition for CaM occurs with ACTH, beta-endorphin, substance P, glucagon, poly(L-arginine), myelin basic protein, troponin I, and histone H2A. Phosphorylation of the last three proteins by the adenosine cyclic 3',5'-phosphate dependent protein kinase diminishes their ability to compete. Phosphorylation of MLCK by the protein kinase gives 0.95 +/- 0.04 and 2.2 +/- 0.4 mol of incorporated 32P in the presence and absence of CaM, respectively. These stoichiometries agree with those recently reported [Conti, M. A. & Adelstein, R. S. (1981) J. Biol. Chem. 256, 3178].     

Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen

Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.     

Interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan

We have examined the interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan, using both equilibrium dialysis and flow dialysis methods. Results obtained by the two procedures were equivalent and indicate that the trp aporepressor binds L-tryptophan with an equilibrium dissociation constant (Kd) of 40 microM at 25 degrees C under standard binding assay conditions (10 mM potassium phosphate, pH 7.4, 0.2 M potassium chloride, 0.1 mM EDTA, 5% glycerol). Molecular sizing of the purified trp aporepressor shows that in the absence of ligand the regulatory protein exists as a dimeric species with greater than 99% purity and an apparent molecular weight of 30,000. Under the storage and assay conditions used, the dimer appears quite stable, and essentially no monomer or higher multimeric species are detected. Analysis of binding data by Scatchard and direct linear plot methods shows two identical and independent ligand-binding sites/native trp aporepressor dimer. When examined as a function of temperature, L-tryptophan binding by trp aporepressor varied over 7-fold (Kd = 28 microM at 6.5 degrees C to Kd = 217 microM at 40 degrees C). At the optimal growth temperature for E. coli (37 degrees C), the dissociation constant was 160 microM for the ligand, L-tryptophan. From the relationship between temperature and L-tryptophan binding by trp aporepressor, the apparent enthalpy change delta H = -10.6 +/- 0.6 kcal mol-1 and the apparent entropy change delta S = -17 +/- 2 cal degree-1 mol-1 were determined.     

Spectrophotometric analysis of the interaction between cytochrome b5 and cytochrome c

The interaction between cytochrome c and the tryptic fragment of cytochrome b5 has been found to produce a difference spectrum in the Soret region with a maximum absorbance at 416 nm. The intensity of this difference has been used to determine the stoichiometry of complex formation and the stability of the complex formed. At pH 7.0 [25 degrees C (phosphate), mu = 0.01 M], the two proteins were found to form a 1:1 complex with an association constant, KA, of 8(3) x 10(4) M-1. The stability of the complex was found to be strongly dependent on ionic strength with KA increasing to 4(3) x 10(6) M-1 at mu = 0.001 M [25 degrees C, pH 7.0 (phosphate)]. Analysis of the dependence of KA on pH from pH 6.5 to 8 demonstrated that this complex is maximally stable between pH 7 and 8 or about midway between the isoelectric points of the two proteins. Analysis of the temperature dependence of KA revealed that formation of the complex between the two proteins is largely entropic in origin with delta Ho = 1 +/- 3 kcal/mol and delta So = 33 +/- 11 eu [pH 7.0 (phosphate), mu = 0.001 M]. This result may be explained either by the model of Clothia and Janin [Clothia, C., & Janin, J. (1975) Nature (London) 256, 705] in terms of extensive solvent reorganization upon complexation or by the model of Ross and Subramanian [Ross, P. D., & Subramanian, S. (1981) Biochemistry 20, 3096] in which the negative enthalpic and entropic contributions of short-range protein-protein interactions are offset by proton release.     

Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem-5.

We have determined the thermodynamic stability and peptide binding affinity of the carboxy-terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal-transduction protein Sem-5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation--at pH 7.3, the protein has a Tm of 73.1 degrees C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm approximately equal to 80 degrees C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more carboxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 degrees C. We have also characterized binding of the domain to 2 different length proline-rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem-5's likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2-fold increase in fluorescence intensity. Both bind with only modest affinities (Kd approximately equal to 30 microM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly-L-proline in a similar fashion.     

Direct determination of the association constant between elongation factor Tu X GTP and aminoacyl-tRNA using fluorescence

We have investigated the formation of the aa-tRNA X EF-Tu X GTP ternary complex spectroscopically by monitoring a fluorescence change that accompanies the association of EF-Tu X GTP with Phe-tRNAPhe-F8, a functionally active analogue of Phe-tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base. With this approach, the protein-nucleic acid interaction could be examined by direct means and at equilibrium. The fluorescence emission intensity of each Phe-tRNAPhe-F8 increased by 36-55% upon association with EF-Tu X GTP, depending on the solvent conditions. Thus, when Phe-tRNAPhe-F8 was titrated with EF-Tu X GTP, the extent of ternary complex formation was determined from the increase in emission intensity. A nonlinear least-squares analysis of the titration data yielded a dissociation constant of 0.85 nM for the ternary complex in 50 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (pH 7.6), 10 mM MgCl2, and 50 mM NH4Cl, at 6 degrees C. The delta H degrees of this interaction, determined by the temperature dependence of Kd, was -16 kcal/mol; the delta S degrees was therefore -16 cal mol-1 deg-1 at 6 degrees C in this buffer. In a more physiological polycation-containing solvent ("polymix"), the Kd was 4.7 nM. The ionic strength dependence of ternary complex formation showed that a minimum of two salt bridges and a substantial nonelectrostatic contribution are involved in the binding of aa-tRNA to EF-Tu. The affinities of unmodified aa-tRNAs for EF-Tu X GTP were determined by their abilities to compete with the fluorescent aa-tRNA for binding to the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analyses of AmpC beta-lactamase through differential stability.

Despite decades of intense study, the complementarity of beta-lactams for beta-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, beta-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free beta-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of beta-lactams for the beta-lactamase AmpC through reversible denaturation of adducts of the enzyme with beta-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 degrees C and a van't Hoff enthalpy of unfolding (deltaH(VH)) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (deltaG(u)H2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (deltaTm = +5.8 degrees C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (deltaTm = +4.9 degrees C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (deltaTm = -3.2 degrees C) and 0.7 kcal/mol (deltaTm = -1.2 degrees C), respectively. These beta-lactams, which share nonhydrogen substituents in the 6(7)alpha position of the beta-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (deltaTm = -1.3 degrees C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the beta-lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for beta-lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other beta-lactam binding proteins as well.     

Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction

A filter retention assay is used to examine the kinetic and equilibrium properties of the interaction between phage R17 coat protein and its 21-nucleotide RNA binding site. The kinetics of the reaction are consistent with the equilibrium association constant and indicate a diffusion-controlled reaction. The temperature dependence of Ka gives delta H = -19 kcal/mol. This large favorable delta H is partially offset by a delta S = -30 cal mol-1 deg-1 to give a delta G = -11 kcal/mol at 2 degrees C in 0.19 M salt. The binding reaction has a pH optimum centered around pH 8.5, but pH has no effect on delta H. While the interaction is insensitive to the type of monovalent cation, the affinity decreases with the lyotropic series among monovalent anions. The ionic strength dependence of Ka reveals that ionic contacts contribute to the interaction. Most of the binding free energy, however, is a result of nonelectrostatic interactions.     

Interaction of calmodulin with the particulate fraction of cardiac muscle

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.     

Reaction of Escherichia coli and yeast photolyases with homogeneous short-chain oligonucleotide substrates

Similar rates have been observed for dimer repair with Escherichia coli photolyase and the heterogeneous mixtures generated by UV irradiation of oligothymidylates [UV-oligo(dT)n, n greater than or equal to 4] or DNA. Comparable stability was observed for ES complexes formed with UV-oligo(dT)n, (n greater than or equal to 9) or dimer-containing DNA. In this paper, binding studies with E. coli photolyase and a series of homogeneous oligonucleotide substrates (TpT, TpTp, pTpT, TpTpT, TpTpT, TpTpTpT, TpTpTpT, TpTpTpT, TpTpTpT) show that about 80% of the binding energy observed with DNA as substrate (delta G approximately 10 kcal/mol) can be attributed to the interaction of the enzyme with a dimer-containing region that spans only four nucleotides in length. This major binding determinant (TpTpTpT) coincides with the major conformational impact region of the dimer and reflects contributions from the dimer itself (TpT, delta G = 4.6 kcal/mol), adjacent phosphates (5'p, 0.8 kcal/mol; 3'p, 1.1 kcal/mol), and adjacent thymine residues (5'T, 0.8 kcal/mol; 3'T, 1.3 kcal/mol). Similar turnover rates (average kcat = 6.7 min-1) are observed with short-chain oligonucleotide substrates and UV-oligo(dT)18, despite a 25,000-fold variation in binding constants (Kd). In contrast, the ratio Km/Kd decreases as binding affinity decreases and appears to plateau at a value near 1. Turnover with oligonucleotide substrates occurs at a rate similar to that estimated for the photochemical step (5.1 min-1), suggesting that this step is rate determining. Under these conditions, Km will approach Kd when the rate of ES complex dissociation exceeds kcat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic characterization of allosteric glycogen phosphorylase inhibitors

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.     

Effects of temperature on cytochrome oxidase activity in solubilized form and in lipid vesicle systems.

Isolated mammalian cytochrome oxidase gave an Arrhenius plot with a break (Tb) at about 20 degrees C when assayed in a medium containing Emasol. The activation energies above and below 20 degrees C were 9.3 (EH) and 18.9 kcal/mol (EL), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature Tt = 40 degrees C), dimyristoyl phosphatidylcholine (DMPC, Tt = 23 degrees C) and dioleoyl phosphatidylcholine (DOPC, Tt = -22 degrees C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36 degrees C with E = 22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with Tb = 23 degrees C. EH and EL were 6.6 and 15.8 kcal/mol, respectively. The plot for the oxidase-DMPC also showed a break (Tb = 26 degrees C) with EH = 6.6 and EL = 26.6 kcal/mol. These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the "hot" and "cold" conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.     

Preferential assembly of the tropomyosin heterodimer: equilibrium studies

Thermal unfolding/refolding studies of the three tropomyosin dimers, alpha alpha, alpha beta, and beta beta, from chicken gizzard muscle were performed to explain the preferential assembly of alpha- and beta-tropomyosin subunits into heterodimers, alpha beta [Lehrer, S. S., & Qian, Y. (1989) J. Biol. Chem. 265, 1134]. Circular dichroism measurements showed that all three dimers unfolded in cooperative reversible transitions with T1/2 = 40.0 degrees C and delta H degrees = 162 kcal/mol for alpha alpha and with T1/2 = 42.6 degrees C and delta H degree = 98 kcal/mol for beta beta at 0.4-0.5 microM concentrations. Fluorescence measurements on pyrenyliodoacetamide-labeled tropomyosin showed that (i) excimer fluorescence decreases in parallel with unfolding of homodimers, (ii) at physiological temperature, heterodimers are formed from micromolar mixtures of homodimers over a period of minutes, and (iii) heterodimers unfold/refold with temperature without appreciable formation of homodimers. To understand the preferential formation of alpha beta, we calculated the concentrations of all species present as a function of temperature for equal total amounts of alpha and beta, using the measured thermodynamic constants of the unfolding/dissociation equilibria for alpha alpha and beta beta. Values for delta H degrees = 225 kcal/mol and T1/2 = 43 degrees C for unfolding of alpha beta at 0.5 microM concentration were obtained from the best fit of the calculations to the measured helical content vs temperature of alpha beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic dissection of the Ezrin FERM/CERMAD interface

ERM (Ezrin-Radixin-Moesin) proteins are key cross-linkers of the plasma membrane and the actin cytoskeleton. They are regulated by the intramolecular association of the N-terminal FERM (band-four point one, Ezrin, Radixin, Moesin) and C-terminal CERMAD (ERM association domain) domains (N/C interaction), which masks the binding surfaces of the domains for other molecules. The N/C interface is characterized by the highly distributed binding of CERMAD through a beta-strand and four alpha-helices to a globular FERM. Though it is a target for multiple regulatory signals, little is known about the dynamics/thermodynamics governing this interface. Recent implications of Ezrin in cancer metastasis have increased the necessity to understand this regulatory switch. In this study, we report residue-specific stabilities of Ezrin CERMAD at the Ezrin N/C interface obtained using hydrogen-deuterium exchange NMR. These stabilities vary across secondary structural elements and identify F583 and L586 as key anchor residues for the most stable element, alphaD. Macroscopic N/C binding energetics, obtained using isothermal titration calorimetry (ITC) reveals a high affinity (Kd =176 nM) enthalpy-driven binding (DeltaH = -26 kcal/mol, TDeltaS = -17 kcal/mol) at 25 degrees C at pH 7 in MES and phosphate buffers. A 10-fold increase in affinity was observed for measurements in acetate buffer, suggesting that an acetate-like molecule might promote the repressed form of the complex, possibly through interaction with the F2 subdomain of FERM, which resembles the acyl-CoA binding protein. In summary, our results have illustrated the dynamic nature of this regulatory interface and provide a foundation for investigating the role of regulatory signals on the stability of this interface.     

Suramin interaction with human alpha-thrombin: inhibitory effects and binding studies

Suramin is a hexasulfonated naphthylurea commonly used as antitrypanosomial drug and more recently for the treatment of malignant tumors. Here we show that suramin binds to human alpha-thrombin inhibiting both the hydrolysis of the synthetic substrate S-2238 (IC50 = 40 microM), and the thrombin-induced fibrinogen clotting (IC50 = 20 microM). The latter is completely reversed by albumin (30 mg mL(-1)) suggesting that, at therapeutic concentrations, suramin is unable to affect alpha-thrombin activity in the plasma. Kinetic analysis showed that suramin acts as a non-competitive inhibitor decreasing Vmax without changing the Km for S-2238 hydrolysis. Calorimetric studies revealed two distinct binding sites for suramin in alpha-thrombin. In addition, circular dichroism studies showed that suramin causes significant changes in alpha-thrombin tertiary structure, without affecting the secondary structure content. Interaction with alpha-thrombin resulted in an increased fluorescence emission of the drug. Complex formation was strongly affected by high ionic strength suggesting the involvement of electrostatic interactions. Altogether our data suggest that part of the biological activities of suramin might be related to alpha-thrombin inhibition at extra-vascular sites.