Showing papers on "Cooperativity published in 1999"

Aqueous Coordination Chemistry of Quinoline-Based Fluorescence Probes for the Biological Chemistry of Zinc

Abstract: Metal-specific fluorescence probes are of increasing importance in understanding the neurobiology and general cell biology of zinc. Several quinoline-based compounds such as TSQ and zinquin have been employed to detect zinc in fluorescence microscopy experiments in vivo; however, the aqueous solution chemistry remains equivocal. In some cases, this family of probes is said to reveal labile pools of Zn(II) inside the cell, yet in other cases, these probes are suggested to remove Zn(II) from tightly bound sites in proteins. Since the binding modes, coordination numbers, and thermodynamics of zinc−zinquin interactions in aqueous solution have not been established, these proposals are difficult to distinguish. Here we show that, under physiological conditions, the various forms of zinquin bind Zn(II) with a high degree of cooperativity forming 2:1 complexes. Potentiometric, UV−visible, and fluorescence methods all yield an overall binding constant of log K = 13.5 under physiological conditions. To put this nu...

Local Control Models of Cardiac Excitation–Contraction Coupling : A Possible Role for Allosteric Interactions between Ryanodine Receptors

Abstract: In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through ryanodine receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation–contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca2+ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation–contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution.

Spontaneous Assembly of Helical Cyanine Dye Aggregates on DNA Nanotemplates

Abstract: 3,3‘-Diethylthiadicarbocyanine (DiSC2(5)) is a symmetrical cationic cyanine dye consisting of two N-ethylated benzothiazole groups linked by a pentamethine bridge. Spectroscopic analysis indicates dimerization of the dye in the presence of duplex DNA sequences consisting of alternating adenine/thymine (A/T) or inosine/cytosine (I/C) residues, based on the following observations: (i) the absorption maximum shifts from 647 to 590 nm, (ii) exciton splitting is observed in the induced circular dichroism spectrum, and (iii) fluorescence from the dye is strongly quenched. Dimerization on I/C, but not G/C sequences indicates that the cyanine dimers insert into the minor groove, a conclusion that is supported by viscometric analysis. Spectroscopic studies with short synthetic oligonucleotide duplexes demonstrate that dimerization is highly cooperative: binding of one dimer greatly facilitates binding of a second dimer. For longer binding sites, this cooperativity leads to the formation of extended helical cyani...

Domains Responsible for Constitutive and Ca2+-Dependent Interactions between Calmodulin and Small Conductance Ca2+-Activated Potassium Channels

Abstract: Small conductance Ca2+-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK α subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements in calmodulin and the α subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the α subunit after calmodulin binding and Ca2+ binding to the α subunit–calmodulin complex. Two-hybrid experiments showed that the Ca2+-independent interaction of calmodulin with the α subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK α subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca2+-independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant calmodulins revealed that channel gating is mediated by Ca2+ binding to the first and second E-F hand motifs in the N-terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca2+-calmodulin-dependent conformational change in the channel α subunits, in which different domains of calmodulin are responsible for Ca2+-dependent and Ca2+-independent interactions. In addition, calmodulin is associated with each α subunit and must bind at least one Ca2+ ion for channel gating. Based on these results, a state model for Ca2+ gating was developed that simulates alterations in SK channel Ca2+sensitivity and cooperativity associated with mutations in CaM.

The Significance of Tetramerization in Promoter Recruitment by Stat5

Abstract: Stat5a and Stat5b are rapidly activated by a wide range of cytokines and growth factors, including interleukin-2 (IL-2). We have previously shown that these signal transducers and activators of transcription (STAT proteins) are key regulatory proteins that bind to two tandem gamma interferon-activated site (GAS) motifs within an IL-2 response element (positive regulatory region III [PRRIII]) in the human IL-2Ralpha promoter. In this study, we demonstrate cooperative binding of Stat5 to PRRIII and explore the molecular basis underlying this cooperativity. We demonstrate that formation of a tetrameric Stat5 complex is essential for the IL-2-inducible activation of PRRIII. Stable tetramer formation of Stat5 is mediated through protein-protein interactions involving a tryptophan residue conserved in all STATs and a lysine residue in the Stat5 N-terminal domain (N domain). The functional importance of tetramer formation is shown by the decreased levels of transcriptional activation associated with mutations in these residues. Moreover, the requirement for STAT protein-protein interactions for gene activation from a promoter with tandemly linked GAS motifs can be relieved by strengthening the avidity of protein-DNA interactions for the individual binding sites. Taken together, these studies demonstrate that a dimeric but tetramerization-deficient Stat5 protein can activate only a subset of target sites. For functional activity on a wider range of potential recognition sites, N-domain-mediated oligomerization is essential.

A new use for the 'wing' of the 'winged' helix-turn-helix motif in the HSF–DNA cocrystal

Abstract: The 1.75 A crystal structure of the Kluyveromyces lactis heat shock transcription factor (HSF) DNA-binding domain (DBD) complexed with DNA reveals a protein-DNA interface with few direct major groove contacts and a number of phosphate backbone contacts that are primarily water-mediated interactions. The DBD, a 'winged' helix-turn-helix protein, displays a novel mode of binding in that the 'wing' does not contact DNA like all others of that class. Instead, the monomeric DBD, which crystallized as a symmetric dimer to a pair of nGAAn inverted repeats, uses the 'wing' to form part of the protein-protein contacts. This dimer interface is likely important for increasing the DNA-binding specificity and affinity of the trimeric form of HSF, as well as for increasing cooperativity between adjacent trimers.

Cooperativity and switching within the three-state model of muscle regulation

Abstract: Thin filament regulation is mediated by the presence of tropomyosin (Tm) and troponin (Tn) on the actin filament Binding of Tm alone induces two states, closed and open (with the equilibrium between them defined by KT), which differ in their affinity for myosin subfragment 1 (S1) Cooperative switching between the states results in characteristic sigmoidal myosin S1 binding curves In the presence of Tn and absence of Ca2+, a third state, blocked, has previously been kinetically shown to be present, leading to the three state model of McKillop and Geeves [(1993) Biophys J 65, 693-701] We have measured equilibrium binding of S1 to phalloidin-stabilized pyrene-actin filaments by monitoring the pyrene fluorescence at 50 nM, a concentration 10-fold lower than previously possible In combination with kinetic studies, we show that the data can be fitted to a modified version of the three-state model with an additional term allowing for a varying apparent cooperative unit size (n) Our results show that the apparent cooperative unit size (n) is dependent upon both the presence of Tn and of Ca2+ Also in the absence of Ca2+, the occupancy of the blocked state (defined by KB) is accompanied by a 2-3-fold reduction in KT These results are discussed in comparison to the Hill model [(1980) Proc Natl Acad Sci USA 77, 3186-3190] and a flexible model of thin filament regulation based upon that of Lehrer et al [(1997) Biochemistry 36, 13449-13455]

Genes regulated cooperatively by one or more transcription factors and their identification in whole eukaryotic genomes.

Abstract: Motivation: The question addressed here is how cooperative interactions among transcription factors (TFs), a very frequent phenomenon in eukaryotic transcriptional regulation, can be used to identify genes that are regulated by one or more TFs with known DNA binding specificities. Cooperativity may be homotypic, involving binding of only one transcription factor to multiple sites in a gene's regulatory region. It may also be heterotypic, involving binding of more than one TF. Both types of cooperativity have in common that the binding sites for the respective TFs form tightly linked 'clusters', groups of binding sites often more closely associated than expected by chance alone. Results: A statistical technique suitable for the identification of statistically significant homotypic or heterotypic TF binding site clusters in whole eukaryotic genomes is presented. It can be used to identify genes likely to be regulated by the TFs. Application of the technique is illustrated with two transcription factors involved in the cell cycle and mating control of the yeast Saccharomyces cerevisiae, indicating that the results obtained are biologically meaningful. This rapid and inexpensive computational method of generating hypotheses about gene regulation thus generates information that may be used to guide subsequent costly and laborious experimental approaches. and that may aid in the assignment of biological functions to putative open reading frames. Availability: Software used for statistical analysis is available from the author upon request.

Cooperative Dynamics in Duplexes of Stacked Hydrogen-Bonded Moieties

Abstract: The self-assembly of two bis-2-ureido-4[1H]-pyrimidinones by eight intermolecular hydrogen bonds results in extremely stable dimers. These dimers exist both in solution and in the solid state as two different isomers. By single-crystal X-ray analyses the structures of both the syn-isomer and the anti-isomer have been determined. In addition, with ROESY experiments the structure of a third isomer is revealed. By a combination of NMR techniques the mutual rate of exchange has been determined. Interestingly, these values are significantly lower than the comparable processes of a single 2-ureido-4[1H]-pyrimidinone. These experiments allowed us to determine the cooperativity in conformational behavior and tautomerization of stable dimers.

p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting

Abstract: DNA binding activity of p53 is crucial for its tumor suppressor function Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53 Our data show that four subunits of p53DBD bend the DNA by 32–36°, whereas wt p53 bends it by 51–57° The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by ≈35° and ≈70°, respectively These results are in accord with molecular modeling studies of the tetrameric complex Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53–DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers This bending is consistent with the inherent sequence-dependent anisotropy of the duplex Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin

p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting (wild-type p53yp53 DNA binding domainyDNA-protein complexyA-tract phasing analysisybent DNA)

Abstract: DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32-36°, whereas wt p53 bends it by 51-57°. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by '35° and '70°, respectively. These results are in accord with molec- ular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53-DNA tetrameric complex is stabilized by DNA overtwisting and bend- ing toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important func- tional implications including possible p53 interactions with chromatin.

Cooperative fluctuations and subunit communication in tryptophan synthase

Abstract: Tryptophan synthase (TRPS), with linearly arrayed subunits R‚‚R, catalyzes the last two reactions in the biosynthesis of L-tryptophan. The two reactions take place in the respective R- and ‚-subunits of the enzyme, and the intermediate product, indole, is transferred from the R -t o the‚-site through a 25 A long hydrophobic tunnel. The occurrence of a unique ligand-mediated long-range cooperativity for substrate channeling, and a quest to understand the mechanism of allosteric control and coordination in metabolic cycles, have motivated many experimental studies on the structure and catalytic activity of the TRPS R2‚2 complex and its mutants. The dynamics of these complexes are analyzed here using a simple but rigorous theoretical approach, the Gaussian network model. Both wild-type and mutant structures, in the unliganded and various liganded forms, are considered. The substrate binding site in the ‚-subunit is found to be closely coupled to a group of hinge residues (‚77-‚89 and ‚376-‚379) near the ‚-‚ interface. These residues simultaneously control the anticorrelated motion of the two ‚-subunits, and the opening or closing of the hydrophobic tunnel. The latter process is achieved by the large amplitude fluctuations of the so-called COMM domain in the same subunit. Intersubunit communications are strengthened in the presence of external aldimines bound to the ‚-site. The motions of the COMM core residues are coordinated with those of the R-‚ hinge residues ‚174-‚179 on the interfacial helix ‚H6 at the entrance of the hydrophobic tunnel. And the motions of ‚H6 are coupled, via helix ‚H1 and RL6, to those of the loop RL2 that includes the R-subunit catalytically active residue Asp60. Overall, our analysis sheds light on the molecular machinery underlying subunit communication, and identifies the residues playing a key role in the cooperative transmission of conformational motions across the two reaction sites.

Subtype-selective positive cooperative interactions between brucine analogs and acetylcholine at muscarinic receptors: functional studies.

Abstract: In radioligand binding studies, it has been reported that brucine, N-chloromethyl brucine, and brucine N-oxide increased the affinity of acetylcholine for M1, M3, and M4 muscarinic receptors, respectively, in a manner consistent with the predictions of the ternary complex allosteric model. We now demonstrate an equivalent ability of these three allosteric agents to modulate the actions of acetylcholine in functional studies in membranes and in whole cells. The enhancing actions of brucine and brucine N-oxide on acetylcholine (ACh) potency at M1 and M4 receptors respectively have been confirmed in guanosine-5'-O-(3-[35S]thio)triphosphate, GTPase, cAMP, and intracellular Ca2+ mobilization assays of function. In general, neither the basal nor the maximally stimulated response to ACh is affected. The subtype-selective allosteric effects of N-chloromethyl brucine on M2 and M3 receptors were shown to be qualitatively and quantitatively the same in guanosine-5'-O-(3-[35S]thio)triphosphate functional assays, in terms of both its affinity and cooperativity with ACh, as those found in binding assays. Neutral cooperativity of N-chloromethyl brucine with ACh on M4 receptor function was also observed, thereby demonstrating its "absolute subtype selectivity": a lack of action at any concentration at M4 receptors and an action at M2 and M3 receptors. The enhancing action of N-chloromethyl brucine on neurogenically released ACh binding at M3 receptors was also detected in whole tissue as an increased contraction of the isolated guinea pig ileum to submaximal electrical stimulation. In conclusion, these functional studies confirm that brucine analogs are allosteric enhancers of ACh affinity at certain muscarinic receptor subtypes.

The ends of tropomyosin are major determinants of actin affinity and myosin subfragment 1-induced binding to F-actin in the open state.

Abstract: Tropomyosin (TM) is thought to exist in equilibrium between two states on F-actin, closed and open [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. Myosin shifts the equilibrium to the open state in which myosin binds strongly and develops force. Tropomyosin isoforms, that primarily differ in their N- and C-terminal sequences, have different equilibria between the closed and open states. The aim of the research is to understand how the alternate ends of TM affect cooperative actin binding and the relationship between actin affinity and the cooperativity with which myosin S1 promotes binding of TM to actin in the open state. A series of rat alpha-tropomyosin variants was expressed in Escherichia coli that are identical except for the ends, which are encoded by exons 1a or 1b and exons 9a, 9c or 9d. Both the N- and C-terminal sequences, and the particular combination within a TM molecule, determine actin affinity. Compared to tropomyosins with an exon 1a-encoded N-terminus, found in long isoforms, the exon 1b-encoded sequence, expressed in 247-residue nonmuscle tropomyosins, increases actin affinity in tropomyosins expressing 9a or 9d but has little effect with 9c, a brain-specific exon. The relative actin affinities, in decreasing order, are 1b9d > 1b9a > acetylated 1a9a > 1a9d >> 1a9a > or = 1a9c congruent with 1b9c. Myosin S1 greatly increases the affinity of all tropomyosin variants for actin. In this, the actin affinity is the primary factor in the cooperativity with which myosin S1 induces TM binding to actin in the open state; generally, the higher the actin affinity, the lower the occupancy by myosin required to saturate the actin with tropomyosin: 1b9d >1a9d> 1b9a > or = acetylated 1a9a > 1a9a > 1a9c congruent with 1b9c.

Energetics and cooperativity of tertiary hydrogen bonds in RNA structure.

Abstract: Tertiary interactions that allow RNA to fold into intricate three-dimensional structures are being identified, but little is known about the thermodynamics of individual interactions. Here we quantify the tertiary structure contributions of individual hydrogen bonds in a “ribose zipper” motif of the recently crystallized Tetrahymena group I intron P4−P6 domain. The 2‘-hydroxyls of P4−P6 nucleotides C109/A184 and A183/G110 participate in forming the “teeth” of the zipper. These four nucleotides were substituted in all combinations with their 2‘-deoxy and (separately) 2‘-methoxy analogues, and thermodynamic effects on the tertiary folding ΔG°‘ were assayed by the Mg2+ dependence of electrophoretic mobility in nondenaturing gels. The 2‘-deoxy series showed a consistent trend with an average contribution to the tertiary folding ΔG°‘ of −0.4 to −0.5 kcal/mol per hydrogen bond. Contributions were approximately additive, reflecting no cooperativity among the hydrogen bonds. Each “tooth” of the ribose zipper (com...

A Conformational Change in the Human Major Histocompatibility Complex Protein HLA-DR1 Induced by Peptide Binding †

Abstract: To investigate a conformational change accompanying peptide binding to class II MHC proteins, we probed the structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms. Peptide binding induced a large decrease in the effective radius of the protein as determined by gel filtration, dynamic light scattering, and analytical ultracentrifugation. It caused a substantial increase in the cooperativity of thermal denaturation and induced alterations in MHC polypeptide backbone structure as determined by circular dichroism. These changes suggest a condensation of the protein around the bound peptide. An antibody specific for ‚58-69 preferentially bound the empty protein, indicating that the peptide-induced conformational change involves the ‚-subunit helical region. The conformational change may have important implications for the mechanisms of intracellular antigen presentation pathways.

A thermodynamic framework and cooperativity in the tertiary folding of a Mg2+-dependent ribozyme.

Abstract: The folding thermodynamics of the catalytic domain from the Bacillus subtilis RNase P RNA is analyzed using circular dichroism and fluorescence spectroscopies, hydroxyl radical protection, and catalytic activity. Folding of this 255-nucleotide ribozyme can be described with three populated species: unfolded (U), intermediate (I), and native (N) states. The U-to-I transition primarily involves secondary structure formation, whereas the I-to-N transition is dominated by tertiary structure formation. The I-to-N transition is highly cooperative as indicated by the coincidence of the four probes applied here. Two isothermal methods are used to determine the stability of the N state relative to the I state at 10 and 37 degrees C. The first method measures the extent of Mg(2+)-induced folding without urea or at constant urea concentrations. The second method measures the extent of urea-induced unfolding at constant Mg(2+) concentrations. Via application of a cooperative binding analysis, the Mg(2+) transition midpoint (K(Mg)), the Hill constant (n), and the urea-dependent surface burial parameter (m value) determined by both methods are identical, indicating that they report the same, reversible folding event. Three conclusions can be drawn from these results. (i) The folding free energy of a Mg(2+)-dependent tertiary RNA structure can be described by the K(Mg) and n parameters according to a cooperative Mg(2+) binding model. (ii) The Hill constant for this tertiary RNA structure probably represents the differential number of Mg(2+) ions bound in the I-to-N transition. (iii) Under physiological conditions, the stability of this large ribozyme is similar to that of small globular proteins.

Magnesium-dependent folding of self-splicing RNA: exploring the link between cooperativity, thermodynamics, and kinetics.

Abstract: Folding of the Tetrahymena self-splicing RNA into its active conformation involves a set of discrete intermediate states. The Mg2+-dependent equilibrium transition from the intermediates to the native structure is more cooperative than the formation of the intermediates from the unfolded states. We show that the degree of cooperativity is linked to the free energy of each transition and that the rate of the slow transition from the intermediates to the native state decreases exponentially with increasing Mg2+ concentration. Monovalent salts, which stabilize the folded RNA nonspecifically, induce states that fold in less than 30 s after Mg2+ is added to the RNA. A simple model is proposed that predicts the folding kinetics from the Mg2+-dependent change in the relative stabilities of the intermediate and native states.

Correlation between the Free Energy of a Channel-Forming Voltage-Gated Peptide and the Spontaneous Curvature of Bilayer Lipids†

Abstract: The aqueous-membrane partitioning of alamethicin, a voltage-gated channel-forming peptide, was measured as a function of the membrane spontaneous curvature. EPR spectroscopy was used to measure the partitioning of the peptide in lipid compositions formed from dioleoylphosphatidylcholine (DOPC) and varied percentages of dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylethanolamine-N-methyl (DOPE-Me), or dioleoylphosphatidylethanolamine-N,N-dimethyl (DOPE-Me2). When the mole fraction of DOPE in mixtures of DOPC/DOPE is increased the binding of alamethicin decreases, and the increase in binding free energy is found to be linearly dependent upon the mole fraction of DOPE in the mixture. Addition of DOPE-Me or DOPE-Me2 also increases the binding free energy, except that the effect is reduced relative to that of DOPE. The free-energy increase per mole fraction of DOPE was found to be 1400 cal/mol, whereas for DOPE-Me and DOPE-Me2 the free-energy changes were 980 and 630 cal/mol, respectively. When the free-energy changes for alamethicin binding are compared with the previously determined spontaneous curvatures for mixtures of DOPC/DOPE and DOPC/DOPE-Me, the free energy of binding is found to be linearly dependent upon the spontaneous curvature of the bilayer lipids. The effects of membrane lipid unsaturation on the partitioning of alamethicin were also measured and are qualitatively consistent with this conclusion. The sensitivity to spontaneous curvature and the cooperativity that is seen in the binding curves for alamethicin are postulated to be a result of a localized thinning of the bilayer promoted by this peptide.