Temperature dependence of switching of the bacterial flagellar motor by the protein CheY(13DK106YW).

The behavior of the bacterium Escherichia coli is controlled by switching of the flagellar rotary motor between the two rotational states, clockwise (CW) and counterclockwise (CCW). The molecular mechanism for switching remains unknown, but binding of the response regulator CheY-P to the motor component FliM enhances CW rotation. This effect is mimicked by the unphosphorylated double mutant CheY13DK106YW (CheY**). To learn more about switching, we measured the fraction of time that a motor spends in the CW state (the CW bias) at different concentrations of CheY** and at different temperatures. From the CW bias, we computed the standard free energy change of switching. In the absence of CheY, this free energy change is a linear function of temperature (. Biophys. J. 71:2227-2233). In the presence of CheY**, it is nonlinear. However, the data can be fit by models in which binding of each molecule of CheY** shifts the difference in free energy between CW and CCW states by a fixed amount. The shift increases linearly from approximately 0.3kT per molecule at 5 degrees C to approximately 0.9kT at 25 degrees C, where k is Boltzmann's constant and T is 289 Kelvin (= 16 degrees C). The entropy and enthalpy contributions to this shift are about -0. 031kT/ degrees C and 0.10kT, respectively.     

Structural insight into the rotational switching mechanism of the bacterial flagellar motor

The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(ΔPAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(ΔPEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor.     

Noise underlies switching behavior of the bacterial flagellum

We report the switching behavior of the full bacterial flagellum system that includes the filament and the motor in wild-type Escherichia coli cells. In sorting the motor behavior by the clockwise bias, we find that the distributions of the clockwise (CW) and counterclockwise (CCW) intervals are either exponential or nonexponential with long tails. At low bias, CW intervals are exponentially distributed and CCW intervals exhibit long tails. At intermediate CW bias (0.5) both CW and CCW intervals are mainly exponentially distributed. A simple model suggests that these two distinct switching behaviors are governed by the presence of signaling noise within the chemotaxis network. Low noise yields exponentially distributed intervals, whereas large noise yields nonexponential behavior with long tails. These drastically different motor statistics may play a role in optimizing bacterial behavior for a wide range of environmental conditions.     

CW and CCW Conformations of the E. coli Flagellar Motor C-Ring Evaluated by Fluorescence Anisotropy

The molecular cascade that controls switching of the direction of rotation of Escherichia coli flagellar motors is well known, but the conformational changes that allow the rotor to switch are still unclear. The signaling molecule CheY, when phosphorylated, binds to the C-ring at the base of the rotor, raising the probability that the motor spins clockwise. When the concentration of CheY-P is so low that the motor rotates exclusively counterclockwise (CCW), the C-ring recruits more monomers of FliM and tetramers of FliN, the proteins to which CheY-P binds, thus increasing the motor's sensitivity to CheY-P and allowing it to switch once again. Motors that rotate exclusively CCW have more FliM and FliN subunits in their C-rings than motors that rotate exclusively clockwise. How are the new subunits accommodated? Does the diameter of the C-ring increase, or do FliM and FliN get packed in a different pattern, keeping the overall diameter of the C-ring constant? Here, by measuring fluorescence anisotropy of yellow fluorescent protein-labeled motors, we show that the CCW C-rings accommodate more FliM monomers without changing the spacing between them, and more FliN monomers at the same time as increasing their effective spacing and/or changing their orientation within the tetrameric structure.     

Thermodynamic properties of ligand binding by monoclonal anti-fluorescyl antibodies

The effects of temperature on the binding of fluorescein by three monoclonal anti-fluorescyl antibodies (4-4-20, 20-19-1, and 20-20-3) were assessed by measurements of affinity constants (Ka) over a temperature range of 2-70 degrees C. Values for Ka were determined from the degree of ligand association by using fluorescence methodology. Curvilinear van't Hoff plots (ln Ka vs. T-1) were observed for all three antibodies, indicating that their standard enthalpy changes (delta Ho) were temperature dependent. This phenomenon was further investigated by plotting the changes in unitary free energy (delta Gu), standard enthalpy (delta Ho), and unitary entropy (delta Su) vs. temperature. Strong temperature dependencies were observed for enthalpy and entropy values, while free energy plots were only weakly dependent on temperature. At low temperatures (4 degrees C), entropy played a major role in the binding of fluorescein by all three antibodies, while enthalpy dominated at higher temperatures. This was a consequence of the negative heat capacity changes (delta Cpo approximately equal to -320 cal K-1 mol-1) observed for these antibodies, which produced a negative trend in both enthalpy and entropy values with increasing temperature. The negative heat capacity values also indicated that the hydrophobic effect was instrumental in the binding of fluorescein. Entropy changes were lower than expected for hydrophobic binding alone, suggesting that other forces were acting to mitigate the hydrophobic effect. One possibility was that the binding of fluorescein acted to restrain vibrational fluctuations in the active-site region, producing negative changes in both heat capacity and entropy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Switching of the Bacterial Flagellar Motor Near Zero Load

Flagellated bacteria, such as Escherichia coli, are able to swim up gradients of chemical attractants by modulating the direction of rotation of their flagellar motors, which spin alternately clockwise (CW) and counterclockwise (CCW). Chemotactic behavior has been studied under a variety of conditions, mostly at high loads (at large motor torques). Here, we examine motor switching at low loads. Nano-gold spheres of various sizes were attached to hooks (the flexible coupling at the base of the flagellar filament) of cells lacking flagellar filaments in media containing different concentrations of the viscous agent Ficoll. The speeds and directions of rotation of the spheres were measured. Contrary to the case at high loads, motor switching rates increased appreciably with load. Both the CW → CCW and CCW → CW switching rates increased linearly with motor torque. Evidently, the switch senses stator-rotor interactions as well as the CheY-P concentration.     

Thermodynamics of anesthetic/protein interactions. Temperature studies on firefly luciferase.

Firefly luciferase is a soluble enzyme which is unusually sensitive to general anesthetics. The inhibition of the highly purified enzyme by three inhalational and three alcohol general anesthetics has been studied as a function of temperature, in the range from 5 to 20 degrees C. Inhibition constants Ki were determined at different temperatures, and van't Hoff plots of ln (Ki) versus reciprocal absolute temperature were found to be linear for all agents. Analysis of these plots gave values for the standard Gibbs free energy, enthalpy and entropy changes for transferring each anesthetic from water to the anesthetic-binding pocket on the protein. The most striking finding was that the enthalpy changes were much more negative for anesthetics binding to the protein than for binding to lipids or simple solvents. Furthermore, amongst the set of anesthetics studied, it was found that increasing potency correlated with favorable enthalpy rather than entropy changes. We discuss our results with respect to the molecular mechanisms underlying general anesthesia.     

Direction of flagellar rotation in bacterial cell envelopes

Cell envelopes with functional flagella, isolated from wild-type strains of Escherichia coli and Salmonella typhimurium by formation of spheroplasts with penicillin and subsequent osmotic lysis, demonstrate counterclockwise (CCW)-biased rotation when energized with an electron donor for respiration, DL-lactate. Since the direction of flagellar rotation in bacteria is central to the expression of chemotaxis, we studied the cause of this bias. Our main observations were: (i) spheroplasts acquired a clockwise (CW) bias if instead of being lysed they were further incubated with penicillin; (ii) repellents temporarily caused CW rotation of tethered bacteria and spheroplasts but not of their derived cell envelopes; (iii) deenergizing CW-rotating cheV bacteria by KCN or arsenate treatment caused CCW bias; (iv) cell envelopes isolated from CW-rotating cheC and cheV mutants retained the CW bias, unlike envelopes isolated from cheB and cheZ mutants, which upon cytoplasmic release lost this bias and acquired CCW bias; and (v) an inwardly directed, artificially induced proton current rotated tethered envelopes in CCW direction, but an outwardly directed current was unable to rotate the envelopes. It is concluded that (i) a cytoplasmic constituent is required for the expression of CW rotation (or repression of CCW rotation) in strains which are not defective in the switch; (ii) in the absence of this cytoplasmic constituent, the motor is not reversible in such strains, and it probably is mechanically constricted so as to permit CCW sense of rotation only; (iii) the requirement of CW rotation for ATP is not at the level of the motor or the switch but at one of the preceding functional steps of the chemotaxis machinery; (iv) the cheC and cheV gene products are associated with the cytoplasmic membrane; and (v) direct interaction between the switch-motor system and the repellent sensors is improbable.     

Coordinated reversal of flagellar motors on a single Escherichia coli cell

An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors.     

Temperature dependence of the kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into half-molecules. A minimum rate at 15 degrees C indicates hydrophobic interaction between the subunits.

The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin to half-molecules has been studied as a function of temperature by using small-angle scattering of X-rays and neutrons. The most striking result of the present investigation is that there is a minimum in reaction rate at about 15 degrees C, and that the rate increases when the temperature is lowered, or raised, from that value. By analyzing the first-order rate constants in terms of transition-state theory it was found that the dissociation is associated with a large and positive change in heat capacity between the activated complex and native alpha 2-macroglobulin (delta CP is in the range 5 to 6 kJ mol-1 K-1). In analogy with pure thermodynamic investigations, where a large change in heat capacity normally is interpreted as a melting of hydrophobic interaction, we therefore propose that hydrophobic interaction is involved in the so-called non-covalent interactions between the subunits of alpha 2-macroglobulin. As a result of the present investigation, it also follows that the free energy of activation delta G has a maximum at about 32 degrees C, whereas the enthalpy of activation delta H and the entropy of activation delta S are zero at about 15 degrees C and 32 degrees C, respectively. These temperatures are slightly dependent upon the concentration of urea and upon whether the reaction is run in a 1H or a 2H medium. Furthermore, from the kinetic point of view, at low temperature the reaction can be characterized as enthalpy driven, whereas at high temperature, it can be characterized as entropy driven.     

Temperature dependence of histidine ionization constants in myoglobin

The standard enthalpy of ionization of six titratable histidines in horse metaquomyoglobin was determined by repeating proton NMR titrations as a function of temperature and using the van't Hoff relationship. It was found that deltaH degrees varies between 16 and 37 kJ mol(-1) in the protein, compared with a value of 29 kJ mol(-1) in free histidine. The standard entropy change was evaluated by combining the enthalpy and free energy changes derived from the pKa values. Although the entropy change could not be precisely and accurately obtained by this method, it could be established that it spans a wide range, from -60 to 0 J K(-1) mol(-1), about the value of -23 J K(-1) mol(-1) for the free histidine. The entropy change was used within the framework of enthalpy-entropy compensation to partition the solvation component from the standard thermodynamic quantities for each of the titrating residues. It was shown that the partitioning of the values in the protein is not readily understood in terms of solvent accessibility or electrostatic interactions. The contribution of solvation effects to the temperature response appeared to be significant only in the case of His-119 and His-48. The standard quantities were also used to explore the energetics of proton binding in the native state at temperatures below the onset of thermal denaturation.     

Physico-chemical aspects of solubility of myosin in aqueous media

Solubility of fish (Labio rohita) myosin has been studied at varying temperatures in presence of various inorganic salts like NaCl, KCl, NaBr, Na2SO4, KI, and organic solutes like sucrose and urea. The effect of pH on the solubility has also been studied both in absence and presence of NaCl. Thermal denaturation temperatures of myosin in presence of NaCl, KCl, NaBr and Na2SO4 were found to be 40 degrees, 40 degrees, 45 degrees and 50 degrees C respectively. Thermodynamic parameters like changes in standard free energy (delta G degrees), enthalpy (delta H degrees) and entropy (delta S degrees) for precipitation of myosin from solution phase to gel phase have been evaluated and the physico-chemical aspects have been critically discussed. The average delta G degrees for gel formation varied only between -30 and -40 kJ/mole of myosin, although the nature of solutes, temperature and folding state of protein have been grossly altered. A compensation effect has also been exhibited from the linear plot of average values of delta H degrees against T delta S degrees for various solutes.     

Analysis of hystereses in force length and force calcium relations

Analysis of the hystereses in the force-length relationship at constant Ca(2+) concentration and in the force-calcium relationship at constant sarcomere length (SL) provides insight into the mechanisms that control cross-bridge (XB) recruitment. The hystereses are related here to two mechanisms that regulate the number of strong XBs: the cooperativity, whereby the number of strong XBs determines calcium affinity, and the mechanical feedback, whereby the shortening velocity determines the duration for which the XBs are in the strong state. The study simulates the phenomena and defines the role of these feedbacks. The model that couples calcium kinetics with XB cycling was built on Simulink software (Matlab). Counterclockwise (CCW) hysteresis, wherein the force response lags behind the SL oscillations, at a constant calcium level, is obtained in the force-length plane when neglecting the mechanical feedback and accounting only for the cooperativity mechanism. Conversely, the force response precedes the SL oscillations, yielding a clockwise (CW) hysteresis when only the mechanical feedback is allowed to exist. In agreement with experimental observations, either CW or CCW hysteresis is obtained when both feedbacks coexist: CCW hystereses are obtained at low frequencies (<3 Hz), and the direction is reversed to CW at higher frequencies (>3 Hz). The cooperativity dominates at low frequencies and allows the muscle to adapt XB recruitment to slow changes in the loading conditions. The changeover frequency from CCW to CW hysteresis defines the velocity limit above which the muscle absorbs rather than generates energy. The hysteresis in the force-calcium relation is conveniently explained by the same cooperativity mechanism. We propose that a single cooperativity mechanism that depends on the number of strong XBs can explain the hystereses in the force-length as well as in the force-calcium relationships.     

Enthalpy, entropy and heat capacity changes induced by binding of calcium ions to cardiac troponin C

Microcalorimetric titrations have been used to study the binding of Ca2+ to cardiac troponin C. Measurements were made both in the presence and in the absence of Mg2+, and at temperatures of 5 degrees, 15 degrees and 25 degrees C. Changes in enthalpy, entropy and heat capacity of troponin C associated with Ca binding have been determined. Cardiac troponin C exhibited a decrease in enthalpy and an increase in entropy associated with Ca binding. Enthalpy changes increased linearly with temperature, indicating that the Ca binding causes negative changes in the heat capacity of troponin C. These results show that the Ca binding causes a strong hydrophobic effect and a tightening of the molecular structure of cardiac troponin C.     

Salmonella typhimurium fliG and fliN mutations causing defects in assembly, rotation, and switching of the flagellar motor.

FliG, FliM, and FliN are three proteins of Salmonella typhimurium that affect the rotation and switching of direction of the flagellar motor. An analysis of mutant alleles of FliM has been described recently (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992). We have now analyzed a large number of mutations in the fliG and fliN genes that are responsible for four different types of defects: failure to assembly flagella (nonflagellate phenotype), failure to rotate flagella (paralyzed phenotype), and failure to display normal chemotaxis as a result of an abnormally high bias to clockwise (CW) or counterclockwise (CCW) rotation (CW-bias and CCW-bias phenotypes, respectively). The null phenotype for fliG, caused by nonsense or frameshift mutations, was nonflagellate. However, a considerable part of the FliG amino acid sequence was not needed for flagellation, with several substantial in-frame deletions preventing motor rotation but not flagellar assembly. Missense mutations in fliG causing paralysis or abnormal switching occurred at a number of positions, almost all within the middle one-third of the gene. CW-bias and CCW-bias mutations tended to segregate into separate subclusters. The null phenotype of fliN is uncertain, since frameshift and nonsense mutations gave in some cases the nonflagellate phenotype and in other cases the paralyzed phenotype; in none of these cases was the phenotype a consequence of polar effects on downstream flagellar genes. Few positions in FliN were found to affect switching: only one gave rise to the CW mutant bias and only four gave rise to the CCW mutant bias. The different properties of the FliM, FliG, and FliN proteins with respect to the processes of assembly, rotation, and switching are discussed.     

Thermodynamic studies on the interaction between sodium n-dodecyl sulphate and histone H2B

The thermodynamic parameters for the interaction of the anionic detergent sodium n-dodecyl sulphate (SDS) with H2B at pH 3.2, 6.4 and 10 have been measured at 27 degrees C and 37 degrees C by equilibrium dialysis to determine the Gibbs energies of detergent binding. The data have been used to obtain the enthalpy of interaction from the temperature dependence of the equilibrium constants from the Van't Hoff relation. The enthalpy of interaction between H2B and SDS is endothermic at pH 3.2, 6.4 and 10. The shapes of the enthalpy curves at pH 3.2 and 10 show some small exothermic contribution which probably indicates folding of H2B. The interactions of H2B-SDS are dominated by the increase in entropy on detergent binding. The larger negative free energy, enthalpy and entropy changes at pH 6.4 are consistent with greater denaturation relative to pH 3.2 and 10.     

Label-free detection of enhanced saccharide binding at pH 7.4 to nanoparticulate benzoboroxole based receptor units

Nanoparticles modified with either 6-amino-1-hydroxy-2,1-benzoxaborolane (3-aminobenzoboroxole) or 3-aminophenylboronic acid were prepared by nucleophilic substitution of a styrene-co-DVB-co-vinylbenzylchloride latex (25 nm). Isothermal titration calorimetry (ITC) was used as a label-free detection method for the analysis of the binding between monosaccharides and these two differently derivatized nanoparticle systems at pH 7.4. Because ITC reveals, thermodynamical parameters such as the changes in enthalpy ΔH, free energy ΔG, and entropy ΔS, possible explanations for the higher binding constants can be derived in terms of entropy and enthalpy changes. In case of the modified nanoparticles, the free energy of binding is dominated by the entropy term. This shows that interfacial effects, besides the intrinsic affinity, lead to a higher binding constant compared with the free ligand. The highest binding constant was found for fructose binding to the benzoboroxole modified nanoparticles: Its value of 1150 M(-1) is twice as high as for the free benzoboroxole and five times as high as with phenylboronic acid or 3-aminophenylboronic acid. In contrast to the binding of fructose to free boronic acids, which is an enthalpically driven process, the binding of fructose to the modified nanoparticles is dominated by the positive entropy term.     

Isatin-enzyme interactions. VII. Mechanism of inhibition of rat kidney alkaline phosphatase.

Isatin has been found to inhibit rat kidney alkaline phosphatase (EC 3.1.3.1). The inhibition is dependent on isatin concentration and is of un-competitive type. The hydrolysis of disodium phenyl phosphate by the enzyme at different temperatures (17--37 degrees C) obeys the Arrhenius equation. Energy of activation in the absence and presence of isatin has been found to be 9.84 and 10.24 kCal/mol. The hyperbolic profile of isatin inhibition; the lowering of both Km and Vmax in the presence of isatin, and, small changes in enthalpy, free energy and entropy in the presence of isatin suggest a non-allosteric un-competitive inhibition of the enzyme.     

Effects of Hofmeister salts on the self-association of glucagon

The trimerization constants of glucagon at pH 10.6 in 0.76 M K2HPO4 have been calculated from circular dichroism data between 5 and 50 degrees C. The free energy, enthalpy, and entropy of transfer have been evaluated from the current results and published data in 0.20 M phosphate. The free energies of transfer are derived completely from an increase in the entropy of transfer, since the enthalpy of transfer is less favorable at all temperatures. These parameters are compared with those of various model groups and compounds: CH2, peptide, methane, ethane, and the 1--13 N-terminal fragments of ribonuclease. The effects of fluoride and chloride on the self-association of glucagon have been compared with that of phosphate at 25 degrees C. These effects are consistent with the binding of approximately one molecule of salt to the trimer and a systematic decrease in the number of water molecules bound to the trimer compared to the monomer for the series K2HPO4, KF, and KCl.     

Mapping heat exchange in an allosteric protein

Nicotinic acetylcholine receptors (AChRs) are synaptic ion channels that spontaneously isomerize (i.e., gate) between resting and active conformations. We used single-molecule electrophysiology to measure the temperature dependencies of mouse neuromuscular AChR gating rate and equilibrium constants. From these we estimated free energy, enthalpy, and entropy changes caused by mutations of amino acids located between the transmitter binding sites and the middle of the membrane domain. The range of equilibrium enthalpy change (13.4 kcal/mol) was larger than for free energy change (5.5 kcal/mol at 25°C). For two residues, the slope of the rate-equilibrium free energy relationship (Φ) was approximately constant with temperature. Mutant cycle analysis showed that both free energies and enthalpies are additive for energetically independent mutations. We hypothesize that changes in energy associated with changes in structure mainly occur close to the site of the mutation, and, hence, that it is possible to make a residue-by-residue map of heat exchange in the AChR gating isomerization. The structural correlates of enthalpy changes are discussed for 12 different mutations in the protein.