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M A Shea

Publications and source records attributed to M A Shea.

At least 19 recordsLinked to original sources
PubMed1995

Discontinuous equilibrium titrations of cooperative calcium binding to calmodulin monitored by 1-D 1H-nuclear magnetic resonance spectroscopy.

Calmodulin binds up to four calcium ions cooperatively in response to cellular signaling events. To understand the functional energetics of calcium activation of calmodulin, it is important to monitor individual Ca(2+)-binding sites and other positions at partial degrees of saturation. This study is the first use of 1-D proton NMR to monitor the equilibrium Ca(2+)-binding properties of calmodulin. Protein concentrations required for NMR experiments (approximately 1 mM) are approximately 1000-fold greater than the Kd values for calcium binding to calmodulin, preventing a direct continuous equilibrium titration of calmodulin. Thus, dialysates of calmodulin in buffers of experimentally determined [Ca2+]free were prepared to conduct discontinuous equilibrium titrations at both 92 and 152 mM KCl. For the C-terminal domain, the normalized area of the delta-protons of Y138 defined calcium binding isotherms. For N-terminal domain resonances (F16C delta H, T26C alpha H, D64C alpha H, and F65C delta H), the calcium-dependent change in chemical shift defined isotherms. These are the first residue-specific studies to monitor the energetics of Ca2+ binding to the N-terminal domain in wild-type holo calmodulin. Calcium binding to both domains appeared cooperative and binding affinity decreased in higher KCl. Isotherms resolved from the side chain resonances of F16 and F65 had a lower median ligand activity and a slightly higher degree of cooperativity than isotherms resolved from the backbone resonances of D64 and T26. Salt-dependent changes in apparent intradomain cooperativity differed for the domains: at higher salt, delta Gc increased for the C-terminal domain while remaining constant or decreasing for the N-terminal domain.

Amino Acid Sequence↗
PubMed1995

Quantitative endoproteinase GluC footprinting of cooperative Ca2+ binding to calmodulin: proteolytic susceptibility of E31 and E87 indicates interdomain interactions.

Calmodulin is the primary eukaryotic intracellular calcium receptor. Cooperative calcium binding to two sites in each of two domains drives large conformational changes that enable it to activate target proteins. An understanding of the molecular mechanism of cooperativity requires determination of the conformational states populated by calmodulin, the intrinsic free energies of binding calcium to four sites, and the nature and degree of intradomain and interdomain interactions. To monitor residue-specific conformational changes within calmodulin as calcium binds, we have developed a new quantitative proteolytic footprinting method using endoproteinase GluC (EndoGluC). Under conditions of very limited proteolysis, cleavage occurred at only five of the sixteen positions possible in calmodulin. The relative abundance of fragments indicated that calcium induced changes in the susceptibility of individual peptide bonds. Quantitative susceptibility profiles were resolved for two positions: E31-L32, in site I in the N-terminal domain, and E87-A88, preceding site III in the C-terminal domain. In apocalmodulin, E87-A88 was susceptible to EndoGluC; calcium binding to sites III and IV caused monotonic protection from proteolysis. The response of E31-L32 was biphasic. In apocalmodulin, it was resistant to cleavage. Susceptibility was induced by calcium binding to sites III and IV, followed by protection induced by calcium binding to sites I and II. This indicated that calmodulin must adopt at least three distinguishable conformations and suggested that the two domains interact. Model-dependent equilibrium constants were resolved from the EndoGluC susceptibility profiles for E31 and E87; they indicated cooperative binding within each domain. Approaches taken to validate this proteolytic footprinting method are described.

Amino Acid Sequence↗
PubMed1994

Bohr effects of the partially-ligated (CN-met) intermediates of hemoglobin as probed by quaternary assembly.

Free energies of quaternary assembly (dimers to tetramers) were determined for the 10 ligation species of CN-methemoglobin in the region of the alkaline Bohr effect (pH 7.0-9.5). Analysis of this database yielded the following principal findings: (1) At each pH, the nine CN-met species exhibit two distinct values of Bohr proton release and Bohr free energy. The two Bohr effects are found to distribute in a fashion that coincides with predictions of a symmetry rule (Ackers et al., 1992), i.e., the first value reflects a "tertiary Bohr effect" arising from ligation within the quaternary T tetramer and a second Bohr effect arises from the quaternary transition (T-->R) which occurs when both dimeric half-molecules acquire at least one ligated subunit. (2) The Bohr effects for CN-met ligation are in good agreement with previously-established Bohr effects for stepwise O2 binding under identical conditions (Chu et al., 1984). (3) In combination with recent studies which show that CN-met species [21] has a quaternary T structure (Daugherty et al., 1991; Doyle & Ackers, 1992; LiCata et al., 1993), the present results show that the "tertiary Bohr effect" within quaternary T exceeds the Bohr effect of dissociated dimers, as suggested by Lee and Karplus (1983). (4) The tertiary Bohr effect is found to account for the pH dependence of tertiary constraint energy, delta Gtc, which "pays" for ligand-binding cooperativity prior to the quaternary (T-->R) switchover. Possible origins of the tertiary Bohr effect and its relationship to the quaternary Bohr effect are considered.

Allosteric Regulation↗
PubMed1991

Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

The 10 ligation species of human cyanomethemoglobin were previously found to distribute into three discrete cooperative free energy levels according to a combinatorial code (i.e., dependent on both the number and configuration of ligated subunits). Analysis of this distribution showed that the hemoglobin tetramer occupies a third allosteric state in addition to those of the unligated (T) and fully ligated (R) species. To determine the nature of the intermediate allosteric state, we have studied the effects of pH, temperature, and single-site mutations on its free energy of quaternary assembly, in parallel with corresponding data on the deoxy (T) and fully ligated (R) species. Results indicate that the intermediate allosteric tetramer has the deoxy (T) quaternary structure. This finding, together with the resolved energetic distribution of the 10 microstates reveals a symmetry rule for quaternary switching--i.e., switching from T to R occurs whenever a binding step creates a tetramer with one or more ligated subunits on each side of the alpha 1 beta 2 intersubunit contact. These studies also reveal significant cooperativity within each alpha 1 beta 1 dimer of the T-state tetramer. The ligand-induced tertiary free energy alters binding affinity within the T structure by 170-fold prior to quaternary switching.

Allosteric Site↗
PubMed1990

Subunit hybridization studies of partially ligated cyanomethemoglobins using a cryogenic method. Evidence for three allosteric states.

Reaction of tetrameric hemoglobin with ligands at the four heme sites yields nine species that have structurally unique combinations of ligated and unligated subunits. Using hemoglobins where the ligated subunits contain cyanomethemoglobin, Smith and Ackers studied the dimer-tetramer assembly reactions in all nine of the partially ligated species (F. R. Smith and G. K. Ackers, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 5347). They found a third assembly free energy in addition to those of unligated hemoglobin and fully ligated cyanomethemoglobin. The observed distribution of the three assembly free energies among the ten species was found to be incompatible with the two-state mechanism of allosteric control (J. Monod, J. Wyman and J. P. Changeaux, J. Mol. Biol. 12 (1965) 81). The results indicated a mechanism of 'combinatorial switching' in which the binding free energies per site change with configuration of occupied sites and not just their number. In this study, we have confirmed the existence of three assembly free energies among the ten ligation species using a cryogenic method (M. Perrella and L. Rossi-Bernardi, Methods Enzymol. 76 (1981) 133). For one of the species we find a different free energy assignment from that reported by Smith and Ackers; for all other species we observe the same assignments as in earlier work. The revised distribution also requires a 'combinatorial' mechanism of allosteric switching among the three states.

Freezing↗
PubMed1986

Energetics of cooperative protein-DNA interactions: comparison between quantitative deoxyribonuclease footprint titration and filter binding.

Using the binding of cI repressor protein to the lambda right and left operators as a model system, we have analyzed the two common experimental techniques for studying the interactions of genome regulatory proteins with multiple, specific sites on DNA. These are the quantitative DNase footprint titration technique [Brenowitz, M., Senear, D. F., Shea, M. A., & Ackers, G. K. (1986) Methods Enzymol. 130, 132-181] and the nitrocellulose filter binding assay [Riggs, A., Suzuki, H., & Bourgeois, S. (1970) J. Mol. Biol. 48, 67-83]. The footprint titration technique provides binding curves that separately represent the fractional saturation for each site. In principle, such data contain the information necessary to determine the thermodynamic constants for local site binding and cooperativity. We show that in practice, this is not possible for all values of the constants in multisite systems, such as the lambda operators. We show how these constants can nevertheless be uniquely determined by using additional binding data from a small number of mutant operators in which the number of binding sites has been reduced. The filter binding technique does not distinguish binding to the individual sites and yields only macroscopic binding parameters which are composite averages of the various local site and cooperativity constants. Moreover, the resolution of even macroscopic constants from filter binding data for multisite systems requires ad hoc assumptions as to a relationship between the number of ligands bound and the filter retention of the complex. Our results indicate that no such relationship exists. Hence, the technique does not permit determination of thermodynamically valid interaction constants (even macroscopic) in multisite systems.

Bacteriophage lambda↗
PubMed1986

"Footprint" titrations yield valid thermodynamic isotherms.

A central issue in gene regulation is the mechanism, and biological function, of the cooperative binding of regulatory protein ligands to specific sites on DNA. To elucidate the physical-chemical basis of these interactions we have developed a thermodynamically rigorous method for conducting DNase I "footprint" (protection) titration experiments. The intrinsic binding constants and also those for cooperative interactions between various sites can be resolved from the individual-site binding curves determined by this technique. Experimental studies of cI-repressor-operator binding have demonstrated that the method provides an accurate representation of the fractional saturation of a binding site. We present individual-site binding curves for a lambda operator with two competent sites that demonstrate the presence of cooperative interactions between the sites. These curves set a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.

Binding Sites↗
PubMed1985

The OR control system of bacteriophage lambda. A physical-chemical model for gene regulation.

A quantitative model has been developed for processes in the bacteriophage lambda that control the switchover from lysogenic to lytic modes of growth. These processes include the interactions of cI repressor and cro proteins at the three DNA sites of the right operator, OR, the binding of RNA polymerase at promoters PR and PRM, the synthesis of cI repressor and cro proteins, and the degradative action of recA during induction of lysis. The model is comprised of two major physical-chemical components: a statistical thermodynamic theory for relative probabilities of the various molecular configurations of the control system; and a kinetic model for the coupling of these probabilities to functional events, including synthesis of regulatory proteins cI and cro. Using independently evaluated interaction constants and rate parameters, the model was found capable of predicting essential physiological characteristics of the system over an extended time. Sufficiency of the model to predict known physiological properties lends credence to the physical-chemical assumptions used in its construction. Several major physiological characteristics were found to arise as "system properties" through the non-linear, time-dependent, feedback-modulated combinations of molecular interactions prescribed by the model. These include: maintenance of the lysogenic state in the absence of recA-mediated cI repressor degradation; induction of lysis and the phenomenon of subinduction; and autogenous negative control of cro. We have used the model to determine the roles, within the composite system, of several key molecular processes previously characterized by studies in vitro. These include: co-operativity in cI repressor binding to DNA; interactions between repressors and RNA polymerase (positive control); and the monomer-dimer association of cI repressor molecules. A major role of cI repressor co-operativity is found to be that of guaranteeing stability of the lysogenic state against minor changes in cI repressor levels within the cell. The role of positive control seems to be that of providing for a peaked, rather than monotonic, dependence of PRM activity on cI repressor level, while permitting PR activity to be a step function. The model correlates an immense body of studies in vivo and in vitro, and it makes testable predictions about molecular phenomena as well as physiological characteristics of bacteriophage lambda. The approach developed in this study can be extended to include more features of the lambda system and to treat other systems of gene regulation.

Allosteric Regulation↗
PubMed1983

Free energy coupling within macromolecules. The chemical work of ligand binding at the individual sites in co-operative systems.

Individual-site binding curves such as those obtainable from techniques of DNase footprinting or nuclear magnetic resonance spectroscopy can be used to monitor structurally localized events within biopolymers. This paper discusses thermodynamic aspects of individual-site ligand binding for co-operative systems where the binding of ligand at a local site is coupled to binding of the same ligand species at other sites within the macromolecule. Individual-site binding isotherms have the following properties. (1) They provide a direct indication of the role played by the particular site in the overall binding reaction. (2) They can be used to determine the energetic contribution of loading the site regardless of the complexity of the system. (3) They can be used to resolve microscopic equilibrium constants and co-operativity constants in cases where the classical isotherm is incapable of such resolution. The microscopic constants bear a complex relation to the chemical work of loading each individual site. For a system with two interacting sites we derive analytical relationships between the individual-site loading energies and the microscopic constants. These relationships prescribe, for any values of the microscopic constants, how the co-operative energy is partitioned between events at the two sites. At fixed ligand activity the binding free energy can be estimated directly from an individual-site isotherm. This quantity, which is also a composite of the microscopic constants, provides a useful measure of site--site interaction. Several examples and applications are discussed for these properties of individual-site binding reactions.

Binding Sites↗
PubMed1982

Quantitative model for gene regulation by lambda phage repressor.

A statistical thermodynamic model has been developed to account for the cooperative interactions of the bacteriophage lambda repressor with the lambda right operator. The model incorporates a general theory for quantitatively interpreting cooperative site-specific equilibrium binding data. Values for all interaction parameters of the model have been evaluated at 37 degrees C, 0.2 M KCl, from results of DNase protection experiments in vitro [A. D. Johnson, B. J. Meyer, & M. Ptashne, Proc. Natl. Acad. Sci. USA (1979) 76, 5061-5065]. With these values, the model predicts repression curves at the divergent promoters PR and PRM that control transcription of genes coding for the regulatory proteins cro and repressor, respectively. At physiological repressor concentrations, repression at PR is predicted to be nearly complete whereas PRM is predicted to remain highly active. The results demonstrate the importance of cooperative interactions between repressor dimers bound to the adjacent operator sites OR1 and OR2 in maintaining a stable lysogenic state and in allowing efficient switchover to the lytic state during induction.

Bacteriophage lambda↗
PubMed1971

Transport as the rate-limiting step in the incorporation of uridine into mengovirus ribonucleic acid in Novikoff rat hepatoma cells.

The incorporation of uridine into the nucleotide pool of actinomycin-treated, mengovirus-infected Novikoff rat hepatoma cells in culture follows simple Michaelis-Menten kinetics, and the apparent V(max) and K(m) values are similar to those for uridine transport by uninfected cells. Incorporation of uridine into mengovirus-specific ribonucleic acid (RNA) also follows Michaelis-Menten kinetics, and the apparent K(m) (about 10 mum) is approximately the same as for uridine transport. Inhibition of uridine transport by the presence of adenosine, persantin, or phenethyl alcohol inhibits simultaneously and to the same extent the incorporation of uridine into the nucleotide pool and into viral RNA, without affecting viral RNA synthesis per se. Phenethyl alcohol, however, also inhibits virus maturation. The inhibition of uridine incorporation into the nucleotide pool and into viral RNA is of the simple competitive type, indicating that transport into the cells is the rate-limiting step in the incorporation of uridine into mengovirus RNA. The results also indicate that treatment with actinomycin D or mengovirus infection does not affect uridine transport.

Biological Transport↗
PubMed1971

Effects of elevated temperatures on mengovirus ribonucleic acid synthesis and virus production in Novikoff rat hepatoma cells.

The production of mengovirus in Novikoff rat hepatoma cells is progressively reduced with an increase in incubation temperature of the cells from 34 to 40 C, in spite of the fact that about the same amounts of single-stranded and double-stranded viral ribonucleic acid (RNA) are synthesized at 34, 37, and 40 C; the rate of overall protein synthesis is as high at 40 C as at 37 C. At 40 C, progeny viral RNA accumulates in an undegraded form without being incorporated into virus particles. The results suggest that virus maturation is preferentially inhibited at supraoptimal temperatures. At 42 C, on the other hand, no viral RNA is produced and no viral RNA polymerase activity is detectable in cell lysates. Failure of infected cells to form viral RNA polymerase at 42 C is probably due to an impairment of protein synthesis since most of the polyribosomes are rapidly lost during incubation at 42 C and the rate of amino acid incorporation into protein is 70% lower at 42 C than at 37 C. When infected cells are shifted from 37 to 42 C during the period of active viral RNA synthesis, viral RNA polymerase activity is rapidly lost from the cells, and viral RNA synthesis ceases within 45 min. In contrast, the RNA polymerase is as active in vitro at 42 C as at 37 C, and the activity is relatively stable at 42 C.

Amino Acids↗