A small molecule inhibits and misdirects assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of -28.0 +/- 2.0 kJ/mol (-6.7 +/- 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was -24.5 +/- 0.9 kJ/mol (-5.9 +/- 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular "wedge" that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.     

Fluorescence study of the thyroxine-dependent conformational changes in human serum transthyretin.

Fluorescence studies of transthyretin (TTR) were conducted to detect structural changes associated with the environment of its two tryptophans, induced by binding of thyroxine (T4). Non-radiative tryptophans relaxation rate has an activation energy of 6.4 kcal/mol for TTR, which is decreased to 4.4 kcal/mol for TTR-T4 complex. The maximum fluorescence wavelength was red-shifted as the excitation wavelength was increased. T4 changed the magnitude of this shift. T4 binding per se changed the emission maximum reflecting different environments of the tryptophans. Double-quenching experiments also showed that T4 produces changes in the tryptophans environments. These findings were interpreted as the result of structural alterations in the protein matrix induced by T4 which contribute in part to explain the negative cooperativity associated with the occupancy of the second binding site.     

The pH-dependent stability of wild-type and mutant transthyretin oligomers

A reduction in pH is known to induce the disassociation of the tetrameric form of transthyretin and favor the formation of amyloid fibers. Using continuum electrostatic techniques, we calculate the titration curves and the stability of dimer and tetramer formation of transthyretin as a function of pH. We find that the tetramer and the dimer become less stable than the monomer as the pH is lowered. The free energy difference is 13.8 kcal/mol for dimer formation and 27 kcal/mol for tetramer formation, from the monomers, when the pH is lowered from 7 to 3.9. Similar behavior is observed for both the wild-type and the mutant protein. Certain residues (namely Glu-72, His-88, His-90, Glu-92, and Tyr-116), play an important role in the binding process, as seen by the considerable pK(1/2) change of these residues upon dimer formation.     

Dimeric procaspase-3 unfolds via a four-state equilibrium process.

We have examined the folding and assembly of a catalytically inactive mutant of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the second transition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).     

Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2

Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the LBD (ligand-binding domain) and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. In the present study, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the LBD of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was used. The potent GluA2 modulator BPAM-97 was used as a reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray structures. The LBD-L483Y-N754S:BPAM-97 complex has a Kd of 5.6?μM (ΔH=-4.9 kcal/mol, -TΔS=-2.3 kcal/mol; where 1?kcal≈4.187?kJ). BPAM-97 was used in a displacement assay to determine a Kd of 0.46?mM (ΔH=-1.2 kcal/mol, -TΔS=-3.3 kcal/mol) for the LBD-L483Y-N754S:IDRA-21 complex. The major structural factors increasing the potency of BPAM-97 over IDRA-21 are the increased van der Waals contacts to, primarily, Met496 in GluA2 imposed by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in the development of drugs against cognitive disorders.     

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)     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.     

Accurate prediction of the binding free energy and analysis of the mechanism of the interaction of replication protein A (RPA) with ssDNA

The eukaryotic replication protein A (RPA) has several pivotal functions in the cell metabolism, such as chromosomal replication, prevention of hairpin formation, DNA repair and recombination, and signaling after DNA damage. Moreover, RPA seems to have a crucial role in organizing the sequential assembly of DNA processing proteins along single stranded DNA (ssDNA). The strong RPA affinity for ssDNA, K(A) between 10(-9)-10(-10) M, is characterized by a low cooperativity with minor variation for changes on the nucleotide sequence. Recently, new data on RPA interactions was reported, including the binding free energy of the complex RPA70AB with dC(8) and dC(5), which has been estimated to be -10 ± 0.4 kcal mol(-1) and -7 ± 1 kcal mol(-1), respectively. In view of these results we performed a study based on molecular dynamics aimed to reproduce the absolute binding free energy of RPA70AB with the dC(5) and dC(8) oligonucleotides. We used several tools to analyze the binding free energy, rigidity, and time evolution of the complex. The results obtained by MM-PBSA method, with the use of ligand free geometry as a reference for the receptor in the separate trajectory approach, are in excellent agreement with the experimental data, with ±4 kcal mol(-1) error. This result shows that the MM-PB(GB)SA methods can provide accurate quantitative estimates of the binding free energy for interacting complexes when appropriate geometries are used for the receptor, ligand and complex. The decomposition of the MM-GBSA energy for each residue in the receptor allowed us to correlate the change of the affinity of the mutated protein with the ΔG(gas+sol) contribution of the residue considered in the mutation. The agreement with experiment is optimal and a strong change in the binding free energy can be considered as the dominant factor in the loss for the binding affinity resulting from mutation.     

Site-specific enthalpic regulation of DNA transcription at bacteriophage lambda OR.

Binding of cI repressor to DNA fragments containing the three specific binding sites of the right operator (OR) of bacteriophage lambda was studied in vitro over the temperature range 5-37 degrees C by quantitative footprint titration. The individual-site isotherms, obtained for binding repressor dimers to each site of wild-type OR and to appropriate mutant operator templates, were analyzed for the Gibbs energies of intrinsic binding and pairwise cooperative interactions. It is found that dimer affinity for each of the three sites varies inversely with temperature, i.e., the binding reactions are enthalpy driven, unlike many protein-DNA reactions. By contrast, the magnitude of the pairwise cooperativity terms describing interaction between adjacently site-bound repressor dimers is quite small. This result in combination with the recent finding that repressor monomer-dimer assembly is highly enthalpy driven (with delta H degrees = -16 kcal mol-1) [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry 30, 7817-7821] indicates that the associative contacts between site-bound repressors that mediate cooperativity are unlikely to be the same as those responsible for dimerization. The intrinsic binding enthalpies for all three sites are negative (exothermic) and nearly temperature-invariant, indicating no heat capacity changes on the scale of those inferred in other protein-DNA systems. However, the three operator sites are affected differentially by temperature: the intrinsic binding free energies for sites OR1 and OR3 change in parallel over the entire range, delta H0OR1 = -23.3 +/- 4.0 kcal mol-1 and delta H0OR3 = -22.7 +/- 1.2 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between D-amino acid oxidase and small molecules.

1. From the standpoint of monomer-dimer equilibrium of hog kidney D-amino acid oxidase [EC 1.4.3.3] and the interaction between the enzyme and small molecules, the effect of pH on the binding of p-aminobenzoate to the monomer and dimer of the enzyme was studied by kinetic methods and spectrophotometric titration. 2. The maximum binding number of p-aminobenzoate to the dimer is two molecules, and there is no interaction between the two active sites of the dimer (i.e., no cooperativity) over the range of pH from 6.5 to 10. 3. The affinity of the dimer for p-aminobenzoate is several times higher than that of the monomer at pH 6.5-10, and consequently p-aminobenzoate induces dimerization in the equilibrium state of D-amino acid oxidase. The interaction energy of two subunits of the dimer is stabilized by the binding of p-aminobenzoate by 1-2 kcal/mole over the pH range studied. 4. The binding sites of the quasi-substrate, p-aminobenzoate, in the dimer and the intersubunit binding site of the dimer are clearly different, because p-aminobenzoate induces dimerization of the enzyme. 5. The pK values of ionizing groups in the free monomer and the free dimer which participate in the binding of the competitive inhibitor, p-aminobenzoate, are approximately the same, 8.7, as determined from the pH dependence of the affinity of the inhibitor for the enzyme. Furthermore, no pK for the enzyme-inhibitor complex in the pH range 6.5-10 was observed. 6. There is no interaction between the two ionizing groups of the dimer during protonation-deprotonation, because a theoretical equation involving no cooperativity between the two ionizing groups in the dimer explains the results well.     

Linkages between the effects of taxol, colchicine, and GTP on tubulin polymerization

A comparative study has been carried out of the effects of taxol on the polymerizations into microtubules of microtubule-associated protein-free tubulin, prepared by the modified Weisenberg procedure, and of the tubulin-colchicine complex into large aggregates. Taxol enhances, to a much greater extent, the stability of microtubules than that of the tubulin-colchicine polymers so that, with highly purified tubulin, assembly into microtubules takes place at 10 degrees C, even in the absence of exogenous GTP. The polymerization of tubulin-colchicine requires both heat and GTP, and the process is reversed by cooling. These results indicate that in both systems polymerization is linked to interactions with taxol and GTP, the interplay of linkage free energies imparting the observed polymer stabilities. In the case of microtubule formation, the linkage free energy provided by taxol binding is approximately -3.0 kcal/mol of alpha-beta-tubulin dimer, whereas this quantity is reduced to approximately -0.5 kcal/mol in tubulin-colchicine, indicating the expenditure of much more binding free energy in the latter case for overcoming unfavorable factors, such as steric hindrance and geometric strain. The difference in the effect of GTP on the two polymerization processes reflects the respective abilities of the bindings of taxol to the two states of tubulin to overcome the loss of the linkage free energy of GTP binding. Analysis of the linkages leads to the conclusions that taxol need not change qualitatively the mechanism of microtubule assembly and that tubulin with the E-site unoccupied by nucleotide should have the capacity to form microtubules, the reaction being extremely weak.     

Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.     

Unfolding of trp repressor studied using fluorescence spectroscopic techniques.

The unfolding properties of the trp repressor of Escherichia coli have been studied using a number of different time-resolved and steady-state fluorescence approaches. Denaturation by urea was monitored by the average fluorescence emission energy of the intrinsic tryptophan residues of the repressor. These data were consistent with a two-state transition from dimer to unfolded monomer with a free energy of unfolding of 19.2 kcal/mol. The frequency response profiles of the fluorescence emission brought to light subtle urea-induced modifications of the intrinsic tryptophan decay parameters both preceding and following the main unfolding transition. The increase of lifetime induced by urea required higher concentrations of urea than the increase in the total intensity described by Gittelman and Matthews [(1990) Biochemistry 29, 7011]. This indicates that the intensity increase has both dynamic and static origins. To assess the effect of tryptophan binding upon repressor stability, and to determine whether repressor oligomerization would be detectable in an unfolding experiment, we examined denaturation profiles of repressor labeled with the long-lived fluorescence probe 5-(dimethylamino)naphthalene-1-sulfonyl (DNS), by monitoring the average rotational correlation time of the probe. These experiments revealed a protein concentration dependent transition at low urea concentrations. This transition was promoted by tryptophan binding. We ascribe this transition to urea-induced dissociation of repressor tetramers. The main unfolding transition of the dimer to unfolded monomer was also observable using this technique, and the free energies associated with this transition were 18.3 kcal/mol in the absence of tryptophan and 24.1 kcal/mol in its presence, demonstrating that co-repressor binding stabilizes the repressor dimer against denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic and thermodynamic framework for assembly of the six-component bI3 group I intron ribonucleoprotein catalyst

The yeast mitochondrial bI3 group I intron RNA splices in vitro as a six-component ribonucleoprotein complex with the bI3 maturase and Mrs1 proteins. We report a comprehensive framework for assembly of the catalytically active bI3 ribonucleoprotein. (1) In the absence of Mg(2+), two Mrs1 dimers bind independently to the bI3 RNA. The ratio of dissociation to association rate constants, k(off)/k(on), is approximately equal to the observed equilibrium K(1/2) of 0.12 nM. (2) At magnesium ion concentrations optimal for splicing (20 mM), two Mrs1 dimers bind with strong cooperativity to the bI3 RNA. k(off)/k(on) is 15-fold lower than the observed K(1/2) of 11 nM, which reflects formation of an obligate intermediate involving one Mrs1 dimer and the RNA in cooperative assembly of the Mrs1-RNA complex. (3) The bI3 maturase monomer binds to the bI3 RNA at almost the diffusion-controlled limit and dissociates with a half-life of 1 h. k(off)/k(on) is approximately equal to the equilibrium K(D) of 2.8 pM. The bI3 maturase thus represents a rare example of a group I intron protein cofactor whose binding is adequately characterized by a one-step mechanism under conditions that promote splicing. (4) Maturase and Mrs1 proteins each bind the bI3 RNA tightly, but with only modest coupling (approximately 1 kcal/mol), suggesting that the proteins interact at independent RNA binding sites. Maturase binding functions to slow dissociation of Mrs1; whereas prior Mrs1 binding increases the bI3 maturase k(on) right to the diffusion limit. (5) At effective concentrations plausibly present in yeast mitochondria, a predominant assembly pathway emerges involving rapid, tight binding by the bI3 maturase, followed by slower, cooperative assembly of two Mrs1 dimers. In the absence of other factors, disassembly of all protein subunits will occur in a single apparent step, governed by dissociation of the bI3 maturase.     

Effects of Zn(II) binding and apoprotein structural stability on the conformation change of designed antennafinger proteins

Ligand-induced conformation change is a general strategy for controlling protein function. In this work, we demonstrate the relationships between ligand binding and conformational stability using a previously designed protein, Ant-F, which undergoes a conformation change upon Zn(II) binding. To investigate the effect of stabilization of the apo structure on the conformation change, we also created a novel protein, Ant-F-H1, into which mutations are introduced to increase its stability over that of Ant-F. The chemical denaturation experiments clarified that apo-Ant-F-H1 is more stable than apo-Ant-F (DeltaDeltaG = -1.28 kcal/mol) and that the stability of holo-Ant-F-H1 is almost the same as that of holo-Ant-F. The Zn(II) binding assay shows that the affinity of Zn(II) for Ant-F-H1 is weaker than that for Ant-F (DeltaDeltaG = 1.40 kcal/mol). A large part of the increased value of free energy in stability corresponds to the decreased value of free energy in Zn(II) binding, indicating that the stability of the apo structure directly affects the conformation change. The denaturation experiments also reveal that Zn(II) destabilizes the conformation of both proteins. From the thermodynamic linkage, Zn(II) is thought to bind to the unfolded state with high affinity. These results suggest that the binding of Zn(II) to the unfolded state is an important factor in the conformational change as well as the stability of the apo and holo structures.     

Linkage between cooperative oxygenation and subunit assembly of cobaltous human hemoglobin.

The thermodynamic linkage between cooperative oxygenation and dimer-tetramer subunit assembly has been determined for cobaltous human hemoglobin in which iron(II) protoporphyrin IX is replaced by cobalt(II) protoporphyrin IX. The equilibrium parameters of the linkage system were determined by global nonlinear least-squares regression of oxygenation isotherms measured over a range of hemoglobin concentrations together with the deoxygenated dimer-tetramer assembly free energy determined independently from forward and reverse reaction rates. The total cooperative free energy of tetrameric cobalt hemoglobin (over all four binding steps) is found to be 1.84 (+/- 0.13) kcal, compared with the native ferrous hemoglobin value of 6.30 (+/- 0.14) kcal. Detailed investigation of stepwise cooperativity effects shows the following: (1) The largest change occurs at the first ligation step and is determined on model-independent grounds by knowledge of the intermediate subunit assembly free energies. (2) Cooperativity in the shape of the tetrameric isotherm occurs mainly during the middle two steps and is concomitant with the release of quaternary constraints. (3) Although evaluation of the pure tetrameric isotherm portrays identical binding affinity between the last two steps, this apparent noncooperativity is the result of a "hidden" oxygen affinity enhancement at the last step of 0.48 (+/- 0.12) kcal. This quaternary enhancement energy is revealed by the difference in subunit assembly free energies of the triply and fully ligated species and is manifested visually by the oxygenation isotherms at high versus low hemoglobin concentration. (4) Cobaltous hemoglobin dimers exhibit apparent anticooperativity of 0.49 (+/- 0.16) kcal (presumed to arise from heterogeneity of subunit affinities).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative binding of R17 coat protein to RNA.

The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays. RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site. The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures. Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA. This system therefore resembles an intermediate state of phage assembly. No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity. Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA.     

Conformational flexibility of neurophysin as investigated by local motions of fluorophores. Relationships with neurohypophyseal hormone binding

Flexibility of various structural domains of neurophysin and neurophysin-neurohypophyseal hormone complexes has been investigated through the fast rotational motion of fluorophores in highly viscous medium. Despite seven intrachain disulfide links, it is shown that some domains of neurophysin remain highly flexible. Dimerization of neurophysin does not affect the structural integrity of the individual subunits, each subdomain being conformationally equivalent within each protomer of the unliganded dimer. The absence of heterogeneous fluorescence anisotropy precludes the existence of a dimer tautomerization equilibrium. Binding of the hormonal ligands to neurophysin dimer promotes a large conformational change over the whole protein structure as assessed by differential alterations of the flexibility-rigidity and intrasegmental interaction properties of domains that do not participate directly to the dimerization/binding areas. The order of free-energy coupling between ligand binding and protein subunit association has been evaluated. Data are consistent with a model in which the first mole of bound ligand stabilizes the dimer by increasing the intersubunit contacts while the second mole of ligand induces most of the described conformational change. Accordingly, the positive cooperativity between the two dimeric binding sites is linked mainly to the binding of the second ligand. The induced structural change is perceived differently by each subunit as assessed by opposite local motions of Tyr49 in each liganded protomer and leads to the formation of a dimeric complex with a global pseudospherical symmetry although containing domains of local asymmetry.     

Thermodynamic analysis of carbon monoxide binding by hemoglobin trout I.

Calorimetric measurements at 25 degrees of the differential heat of CO binding by hemoglobin trout I have been examined together with the CO binding isotherms for the protein at 4 degrees and 20 degrees. Simultaneous treatment of these data sets by a statistically rigorous technique permits evaluation of all the thermodynamic parameters for both the Adair and the Monod, Wyman, Changeux (MWC) models. The results show the details of the unusual temperature dependent cooperativity which this hemoglobin exhibits. In the Adair formalism the increasingly favorable free energy change for successive steps of ligand binding are nearly linearly paralleled by increasingly negative enthalpy changes for these steps. This causes the enhanced cooperativity observed as the temperature is decreased. For the MWC case, lowering the temperature increases the stability of the unligated T state relative to the unligated R state since the enthalpy of the T leads to R transition is 29.4 kcal mol-1. Simultaneously, the favorability of ligating R forms relative to T is enhanced since R form ligation is 14.1 kcal (mol CO)-1 more exothermic than that of T. The balance between these opposing effects is to increase ligand binding cooperativity at low temperatures. The predicted temperature dependence of the Hill coefficient for the MWC and Adair models is identical at low and intermediate temperatures, but, interestingly, would show a strong divergence at high temperatures where negative cooperativity is suggested for the Adair case and positive cooperativity for the MWC case.