The influence of structural variations in the F- and FG-helix of the β-subunit modified oxyHb-NES on the heme structure detected by resonance Raman spectroscopy

The dispersion of the depolarization ratio of two prominent Raman lines (1,375 cm^–1 and 1,638 cm^–1) of oxyhemoglobin-N-ethyl succinimide have been examined for pH values between pH=6.0 and 8.5. Both exhibit a significant pH dependence. Calculation of the Raman tensor in terms of a fifth-order time dependent theory provides information about the pH-dependence of parameters reflecting symmetry classified distortions of the prosthetic heme group. To correlate these distortions with the functional properties of the molecule the following protocol was used: 1) An allosteric model suggested by Herzfeld and Stanley (1974) has been applied to O₂-binding curves measured at different pH values between 6.5 and 9.0. From this calculation one obtains both, the energy differences between different molecular conformations and the equilibrium constants of oxygen and proton binding. 2) A titration model was formulated relating each conformation of a molecule to a distinct set of distortion parameters of the heme group. 3) The distortion parameters resulting from the analysis of our Raman data were assigned as an effective value due to incoherent superposition of the distortion parameters related to the different titration states. The application of this procedure yields an excellent reproduction of the pH-dependent effective distortion parameters of both Raman lines investigated. It is shown that the protonation of two tertiary effector groups located in the -subunits affect the symmetry of the heme in a contrary manner: the protonation of a His-residue (pK=8.2, probably His(FG4)) causes a symmetric position of the proximal imidazole thus lowering the perturbations of the heme core. Further it influences the interaction between amino acid residues of the heme cavity and pyrrole side chains (probably Val (FG5)-vinyl (pyrrole 3) thus causing a decrease of the distortions related to the peripheral part of the heme. In contrast, the protonation of Lys (EF6) causes a tilt position of the proximal imidazole and an increase of asymmetric perturbations of the heme core, whereas the interaction between the pyrrole side chains and the heme cavity is weakened. Our results are consistent with stereochemical predictions of Moffat (1971) concerning the existence of an H-bond between His(FG4) and Cys(F9).Abbreviations DPR depolarization ratio - EP excitation profile - HbA human hemoglobin - oxyHb oxyhemoglobin - NEM N-ethyl-maleimide - NES N-ethyl-succimide - BME Bis (N-maleimidodimethyl)ether     

Ligand binding and allostery can emerge simultaneously

A heterotropic allosteric effect involves an effector molecule that is distinct from the substrate or ligand of the protein. How heterotropic allostery originates is an unanswered question. We have previously created several heterotropic allosteric enzymes by recombining the genes for TEM1 beta-lactamase (BLA) and maltose binding protein (MBP) to create BLAs that are positively or negatively regulated by maltose. We show here that one of these engineered enzymes has approximately 10(6) M(-1) affinity for Zn(2+), a property that neither of the parental proteins possesses. Furthermore, Zn(2+) is a negative effector that noncompetitively switches off beta-lactam hydrolysis activity. Mutagenesis experiments indicate that the Zn(2+)-binding site does not involve a histidine or a cysteine, which is atypical of natural Zn(2+)-binding sites. These studies also implicate helices 1 and 12 of the BLA domain in allosteric signal propagation. These results support a model for the evolution of heterotropic allostery in which effector affinity and allosteric signaling emerge simultaneously.     

Benzodiazepine ligands can act as allosteric modulators of the Type 1 cholecystokinin receptor

The cholecystokinin (CCK₁) receptor is a G protein-coupled receptor important for nutrient homeostasis. The molecular basis of CCK-receptor binding has been debated, with one prominent model suggesting occupation of the same region of the intramembranous helical bundle as benzodiazepines. Here, we used a specific assay of allosteric ligand interaction to probe the mode of binding of devazepide, a prototypic benzodiazepine ligand. Devazepide elicited marked slowing of dissociation of pre-bound CCK, only possible through binding to a topographically distinct allosteric site. This effect was disrupted by chemical modification of a cysteine in the benzodiazepine-binding pocket. Application of an allosteric model to the equilibrium interaction between a series of benzodiazepine ligands and CCK yielded quantitative estimates of each modulator’s affinity for the allosteric site, as well as the degree of negative cooperativity for the interaction between occupied orthosteric and allosteric sites. The allosteric nature of benzodiazepine binding to the CCK₁ receptor provides new opportunities for small molecule drug development.     

Sodium ions as the effector of catalytic action of alpha-thrombin

A process of thrombin interaction with synthetic and natural substrates in the presence of Na+ ions has been analyzed in the survey. Molecular bases of this interaction have been presented, interrelation between the structure and function of thrombin has been noted; the nature of the unique site of its active centre which determines high thrombin affinity for the substrates and increase of its catalytic activity defined by the term of "specificity to univalent cations" have been considered in detail. Na+ ions play the role of allosteric effector in realization of two informational states of thrombin which penform, respectively, two fundamental and competing functions in the process of hemostasis. The molecular basis of the process of Na+ binding with thrombin is rather simple and depends only on the single site which importance for the enzyme function is marked by numerous investigations of a number of authors, and it is shown that Na(+)-binding site is distributed in the other zone of thrombin molecule as compared to exosites I and II, which do not take part in Na(+)-binding and allosteric transduction. Considerable attention was given to conformational conversions of a thrombin molecule caused by Na+ ions binding. It was shown that the transition slow <--> fast of the enzyme forms leads to formation of the ion pair Arg-187: Asp-222, optimal orientation of Asp-189 and Ser-195 for binding of substrates and considerable shift of the lateral chain Glu-192 determined by the disturbance of the lattice of water molecules which connects Na(+)-binding site with aminoacid Ser-195 of the active centre of the enzyme. New data have been presented which indicate that the changes in the lattice of water molecules and allosteric nucleus of Na(+)-binding site of the enzyme are the basic link of raising the affinity between the thrombin and substrate and mechanism of the enzyme activation by Na(+)-ions. The survey touches some problems of creation of allosteric inhibitors of thrombin which can take essential effect on Na(+)-binding site and favor stabilization of the anticoagulant slow-form of thrombin, and of enzyme rational mutants with selective specificity in respect of protein C which display effective and safe anticoagulant and antithrombotic effects in vivo.     

Mechanism of the control of dioxygen binding in a dimeric cobalt-substituted insect hemoglobin. Resonance Raman evidence for cobalt-axial-ligand bond changes

Resonance Raman spectroscopy has been used to investigate the allosteric control mechanism for O2 binding in a cobalt-substituted dimeric insect hemoglobin (CTT II), which exhibits a large Bohr effect due to a pH-induced transition between two ligand affinity states. Substitution of cobalt for iron in CTT II does not modify the Bohr effect, but permits the resonance enhancement (hence the detection) of Raman lines corresponding to the vibrations of the axial ligand-cobalt bonds. Using 16O2/18O2 isotope substitution the O-O and Co-O2 stretching and the Co-O-O bending mode have been assigned to the two affinity states of this hemoglobin: v (O-O) changes from 1152 cm-1 (pH 5.5; t conformation) to about 1125 cm-1 (pH 9.5, r conformation), v (Co-O2) from 512 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) and delta (Co-O-O) from 378 cm-1 (pH 5.5) to 390 cm-1 (pH 9.5). The Co-N epsilon (His) stretching mode has also been detected changing from 313 cm-1 (pH 5.5) to 307 cm-1 (pH 9.5). For the first time, reciprocal behaviour between the Co-N epsilon and Co-O2 bonds and between the Co-O2 and the O-O bonds in an allosteric hemoglobin are demonstrated. Furthermore, the pH sensitivity of a vinyl bending mode in the range of 411-415 cm-1 has been investigated and shown also to reflect the t in equilibrium with r conformation transition.     

An investigation of the nature of Bohr,Root, and haldane effects inOctopus dofleini hemocyanin

1. The pH dependence of Octopus dofleini hemocyanin oxygenation is so great that below pH 7.0 the molecule does not become fully oxygenated, even in pure O2 at 1 atm pressure. However, the curves describing percent oxygenation as a function of PO2 appear to be gradually increasing in oxygen saturation, rather than leveling out at less than full saturation. Hill plots indicate that at pH 6.6 and below the molecule is stabilized in its low affinity conformation. Thus, the low saturation of this hemocyanin in air is due to the very large Bohr shift, and not to the disabling of one or more functionally distinct O2 binding sites on the native molecule. 2. Experiments in which pH was monitored continuously while oxygenation was manipulated in the presence of CO2 provide no evidence of O2 linked binding of CO2. While CO2 does influence O2 affinity independently of pH, its effect may be due to high levels of HCO3- and CO3-, rather than molecular CO2, and it may entail a lowering of the activities of the allosteric effectors Mg2+ and Ca2+.     

Leukocyte function-associated antigen 1-mediated adhesion stability is dynamically regulated through affinity and valency during bond formation with intercellular adhesion molecule-1

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(2) integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.     

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O(2)-affinities (K(T)=0.0085mmHg(-1) and K(R)=0.011mmHg(-1)) and thus a very low cooperativity (K(R)/K(T)=1.3 and L(0)=2.4). (1)H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O(2)-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, (1)H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α(1)β(2)- (or α(2)β(1)-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15-25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O(2), the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O(2), but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O(2) (P(O2)) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P(O2) of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α(1)G- and β(1)G-helices, and the other weaker binding site causes the R→T transition without release of O(2). The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α(1)β(1) (or α(2)β(2)) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α(1)α(2)- (or β(1)β(2)-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O(2)-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb - [the deoxy-state]=[the T-quaternary structure]=[the low O(2)-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O(2)-affinity state] and the O(2)-affiny of Hb being regulated by the T/R-quaternary structural transition - are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Heme structural perturbation of PEG-modified horseradish peroxidase C in aromatic organic solvents probed by optical absorption and resonance Raman dispersion spectroscopy

The heme structure perturbation of poly(ethylene glycol)-modified horseradish peroxidase (HRP-PEG) dissolved in benzene and toluene has been probed by resonance Raman dispersion spectroscopy. Analysis of the depolarization ratio dispersion of several Raman bands revealed an increase of rhombic B(1g) distortion with respect to native HRP in water. This finding strongly supports the notion that a solvent molecule has moved into the heme pocket where it stays in close proximity to one of the heme's pyrrole rings. The interactions between the solvent molecule, the heme, and the heme cavity slightly stabilize the hexacoordinate high spin state without eliminating the pentacoordinate quantum mixed spin state that is dominant in the resting enzyme. On the contrary, the model substrate benzohydroxamic acid strongly favors the hexacoordinate quantum mixed spin state and induces a B(2g)-type distortion owing to its position close to one of the heme methine bridges. These results strongly suggest that substrate binding must have an influence on the heme geometry of HRP and that the heme structure of the enzyme-substrate complex (as opposed to the resting state) must be the key to understanding the chemical reactivity of HRP.     

Iron-histidine stretching vibration in the deoxy state of insect hemoglobins with different O2 affinities and Bohr effects

Resonance Raman spectroscopy has been employed to detect the iron-proximal histidine stretching mode in deoxyhemoglobins from insect larvae of Chironomus thummi thummi (CTT). With the excitation of 413.1 nm, we observe a sharp and intense line in the 220-224 cm-1 region. The assignment of this line to the Fe-N epsilon (His) stretching mode was made on the basis of a 3-cm-1 shift upon 57Fe/54Fe isotope substitution. The Fe-N epsilon (His) vibration is used to monitor the possible changes in the Fe-N epsilon (His) bond strength (hence bone length) in the deoxy state of the monomeric (CTT I, III, and IV) and dimeric (CTT II) insect hemoglobins. As these hemoglobins differ in O2 affinity, off-rate and on-rate constants, and in the Bohr effect, they are excellent model systems for investigating the mechanism of protein control of the heme reactivity. Some of these hemoglobins (CTT III, IV, and II) are allosteric, exhibiting two interconvertible conformational states with high and low O2 affinity at high and low pH, respectively. The Fe-N epsilon (His) stretching frequency does not correlate with the O2 affinity, the on-rate and the off-rate constants for different hemoglobins, for different conformational states, and for modified hemoglobins with different heme peripheral groups. This vibrational mode is insensitive to deuteration of the heme vinyl groups. It is important to note that the Fe-N epsilon (His) bonds in the high pH (high-affinity) and the low pH (low-affinity) states are identical. This implies that the O2 molecule, prior to binding, "sees" identical binding sites. Thus, the difference in free energy changes upon O2 binding is manifested only in the oxy form.     

Characterization of Ca2+-binding proteins from Ehrlich ascites tumor cell cytoplasm and their binding to membranes

A set of proteins in the 33-37 kDa range have been isolated from the cytoplasm of the Ehrlich ascites tumor cell. The proteins are characterized by their Ca2+-dependent binding to cell membranes. This property has been used for isolation of the proteins by Ca2+-dependent affinity binding to inside-out vesicles of the human red cell membrane. The proteins display Ca2+-binding properties as shown by gel-filtration studies. The Ca2+-dependent binding of the 33 and 34 kDa proteins to red cell membranes was studied after labelling of the proteins with tritium by reductive methylation. The average number of Ca2+ bound per protein molecule was 4.8 with a Kd of 3.4.10(-4) M Ca2+. The proteins are distinct from most other Ca2+-binding proteins of comparable molecular weights by not incorporating phosphate.     

Molecular structure,spectroscopic (FT-IR,FT-Raman,UV–vis), NBO,thermochemistry analysis of bis(thiourea)zinc(II) chloride crystals

The experimental and theoretical studies on the molecular structure and vibrational spectra of bis(thiourea)zinc(II) chloride (BTZC) crystals were investigated. The Fourier transform infrared, Fourier transform Raman and UV–vis spectra of BTZC were recorded. The molecular geometry and vibrational frequencies of BTZC in the ground state were calculated by using B3LYP with LANL2DZ as basis set. Comparison of the observed structural parameters of BTZC with single-crystal X-ray studies yields a good agreement. Vibrational analysis of the simultaneous IR and Raman activation of the Zn–Cl stretching mode in the molecule provides the evidence for the charge transfer interaction taking place within the molecule. The energy and oscillator strength are calculated by time-dependent density functional theory. The simulated spectra satisfactorily coincide with the experimental spectra.     

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10 Å from the active site, while epitope β is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a β epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.     

Novel mechanism for high-altitude adaptation in hemoglobin of the Andean frog Telmatobius peruvianus

In contrast to birds and mammals, no information appears to be available on the molecular adaptations for O(2) transport in high-altitude ectothermic vertebrates. We investigated Hb of the aquatic Andean frog Telmatobius peruvianus from 3,800-m altitude as regards isoform differentiation, sensitivity to allosteric cofactors, and primary structures of the alpha- and beta-chains, and we carried out comparative O(2)-binding measurements on Hb of lowland Xenopus laevis. The three T. peruvianus isoHbs show similar functional properties. The high O(2) affinity of the major component results from an almost complete obliteration of chloride sensitivity, which correlates with two alpha-chain modifications: blockage of the NH(2)-terminal residues and replacement by nonpolar Ala of polar residues Ser and Thr found at position alpha131(H14) in human and X. leavis Hbs, respectively. The data indicate adaptive significance of alpha-chain chloride-binding sites in amphibians, in contrast to human Hb where chloride appears mainly to bind in the cavity between the beta-chains. The findings are discussed in relation to other strategies for high-altitude adaptations in amphibians.     

Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes

Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3',5'-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches.     

Exploring allosteric coupling in the α-subunit of Heterotrimeric G proteins using evolutionary and ensemble-based approaches

Background  

Allosteric coupling, which can be defined as propagation of a perturbation at one region of the protein molecule (such as ligand binding) to distant sites in the same molecule, constitutes the most general mechanism of regulation of protein function. However, unlike molecular details of ligand binding, structural elements involved in allosteric effects are difficult to diagnose. Here, we identified allosteric linkages in the α-subunits of heterotrimeric G proteins, which were evolved to transmit membrane receptor signals by allosteric mechanisms, by using two different approaches that utilize fundamentally different and independent information.     

Global allostery model of hemoglobin. Modulation of O(2) affinity,cooperativity, and Bohr effect by heterotropic allosteric effectors

The O(2) equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O(2) affinity. The O(2) affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O(2) carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O(2)-linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model. However, the interactions with allosteric effectors provide such "inert" hemoglobin unprecedented magnitudes of functional diversities not only of physiological relevance but also of extreme nature, by which hemoglobin can behave energetically beyond what can be explained by the Monod-Wyman-Changeux/Perutz model. Thus, the heterotropic effector-linked tertiary structural changes rather than the homotropic ligation-linked T/R quaternary structural transition are energetically more significant and primarily responsible for modulation of functions of hemoglobin.     

Positive heterotropic allosteric regulators of dihydropyridine binding increase the Ca2+ affinity of the L-type Ca2+ channel. Stereoselective reversal by the novel Ca2+ antagonist BM 20.1140.

The alpha 1-subunit of the voltage-dependent L-type Ca2+ channel has distinct, allosterically coupled binding domains for drugs from different chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, diphenylbutylpiperidines). (-)-BM 20.1140 (ethyl-2,2-di-phenyl-4-(1-pyrrolidino)-5-(2-picolyl)- oxyvalerate) is a novel Ca2+ channel blocker which potently stimulates dihydropyridine binding (K0.5 = 2.98 nM) to brain membranes. This property is shared by (+)-cis-diltiazem, (+)-tetrandrine, fostedil and trans-diclofurime, but (-)-BM 20.1140 does not bind in a competitive manner to the sites labeled by (+)-cis-[3H]diltiazem. (+)-cis-Diltiazem and (-)-BM 20.1140 have differential effects on the rate constants of dihydropyridine binding. (+)-BM 20.1140 reverses the stimulation of the positive allosteric regulators (pA2 value for reversal of (-)-BM 20.1140 stimulation = 7.4, slope 0.72). The underlying molecular mechanism of the potentiation of dihydropyridine binding has been clarified. The K0.5 for free Ca2+ to stabilize a high affinity binding domain for dihydropyridines on purified L-type channels from rabbit skeletal muscle is 300 nM. (+)-Tetrandine (10 microM) increases the affinity 8-fold (K0.5 for free Ca2+ = 30.1 nM) and (+)-BM 20.114 (10 microM) inhibits the affinity increase (K0.5 for free Ca2+ = 251 nM). Similar results were obtained with membrane-bound Ca(2+)-channels from brain tissue which have higher affinity for free Ca2+ (K0.5 for free Ca2+ = 132 nM) and for dihydropyridines compared with skeletal muscle. It is postulated that the dihydropyridine and Ca(2+)-binding sites are interdependent on the alpha 1-subunit, that the different positive heterotropic allosteric regulators (by their differential effects on Ca2+ rate constants) optimize coordination for Ca2+ in the channel pore and, in turn, increase affinity for the dihydropyridines.     

Analysis of oxygen binding to Panulirus japonicus hemocyanin. The effect of divalent cations on the allosteric transition

The effects of H+ and divalent cations on the O2 equilibrium of hexameric hemocyanin from a spiny lobster, Panulirus japonicus, were examined. The hemocyanin showed the normal Bohr effect. When divalent cations were removed by EDTA treatment, the protein showed a fivefold increase in the O2 affinity and a considerable decrease in the cooperativity. Several cooperativity models were tested for the conformity with the observed O2-binding isotherms by the least-square curve fitting. Among the models examined, the three-state concerted model was found to be most consistent with the results. It was postulated that in the absence of divalent cations deoxyhemocyanin is mainly in the intermediate-affinity state. The arthropod hemocyanins were found to be classifiable into two groups according to their functional responses to the divalent cations. It was suggested that the cations act differently on the allosteric transitions of the two groups of hemocyanins.