Continuum model of voltage-dependent gating. Macroscopic conductance, gating current, and single-channel behavior.

It is assumed that the conformational change of the voltage-gated channel is continuous, characterized by movement along a generalized one-dimensional reaction coordinate, x, varying from 0 to 1. This large conformational change is coupled to the movement of most of the gating charge. Superimposed on this large movement is a smaller, very fast conformational change that opens or closes the channel. The large conformational change perturbs the channel so that opening is favored near x = 1 and closing is favored near x = 0. The movement along the x axis is described by a generalized Nernst-Planck equation, whereas the open-close transition is modeled as a discrete reaction-rate process. The macroscopic conductance, gating current, and single-channel behavior of a simple, linearized version of the model is described. Although the model has only seven adjustable constants (about the same as would be required for a conventional three-state model), it can mimic the behavior of the delayed rectified K+ channel with 12 or more closed states. The single-channel behavior of the model can have bursts of rapid openings and closings, separated by long closed times. If the conformational change is assumed to correspond to the rotation and translation of charged helices, then this model can be used to estimate the effective rotational diffusion coefficient of the helix. Such calculations for the delayed rectifier K+ channel indicate that the motion must be very restricted.     

Stretched-Exponential Analysis of Heat-Induced Aggregation of Apo-Concanavalin A

The kinetics of heat-induced aggregation of apo-concanavalin A (aConA) was investigated as a function of temperature and protein concentration by circular dichroism and turbidity. Heat-induced aggregation, as well as conformational change, of aConA was fitted to stretched-exponential equations. The exponent of the conformational change maintained 0.5 despite the protein concentration and temperature, indicating the presence of a common intermediate during the conformational change. After the process, aggregates grew with increasing temperature and initial protein concentration. The reaction order of aggregation was 1.5, indicating that the rate-limiting steps of aConA aggregation involve both conformational change and aggregation.     

Dependence of tyrosine oxidation in highly oxidizing bacterial reaction centers on pH and free-energy difference

The pH and temperature dependences of tyrosine oxidation were measured in reaction centers from mutants of Rhodobacter sphaeroides containing a tyrosine residue near a highly oxidizing bacteriochlorophyll dimer. Under continuous illumination, a rapid increase in the absorption change at 420 nm was observed because of the formation of a charge-separated state involving the oxidized dimer and reduced primary quinone, followed by a slow absorption decrease attributed to tyrosine oxidation. Both the amplitude and rate of the slow absorption change showed a pH dependency, indicating that, at low pH, the rate of tyrosine oxidation is limited by the transfer of the phenolic proton to a nearby base. Below 17 degrees C, the rate of the slow absorption change had a strong exponential dependence on the temperature, indicating a high activation energy. At higher pH and temperature, the overall rate of tyrosyl formation appears to be limited by a proposed conformational change in the reaction center that is also observed in reaction centers that do not undergo tyrosine oxidation. The yield of tyrosyl formation measured using electron paramagnetic resonance spectroscopy decreased significantly at 4 degrees C compared to 20 degrees C and was lower at both temperatures in mutants expected to have a slightly smaller driving force for tyrosyl formation.     

Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: role of local internal viscosity in protein dynamics

Upon activation of trypsinogen four peptide segments flanked by hinge glycine residues undergo conformational changes. To test whether the degree of conformational freedom of hinge regions affects the rate of activation, we introduced amino acid side chains of different characters at one of the hinges (position 193) and studied their effects on the rate constant of the conformational change. This structural rearrangement leading to activation was triggered by a pH-jump and monitored by intrinsic fluorescence change in the stopped-flow apparatus. We found that an increase in the size of the side chain at position 193 is associated with the decrease of the reaction rate constant. To analyze the thermodynamics of the reaction, temperature dependence of the reaction rate constants was examined in a wide temperature range (5-60 degrees C) using a novel temperature-jump/stopped-flow apparatus developed in our laboratory. Our data show that the mutations do not affect the activation energy (the exponential term) of the reaction, but they significantly alter the preexponential term of the Arrhenius equation. The effect of solvent viscosity on the rate constants of the conformational change during activation of the wild type enzyme and its R193G and R193A mutants was determined and evaluated on the basis of Kramers' theory. Based on this we propose that the reaction rate of this conformational transition is regulated by the internal molecular friction, which can be specifically modulated by mutagenesis in the hinge region.     

Studies of the role of catalytic and conformational metals in producing enzymatic activity in yeast enolase

Spectrophotometric titrations of yeast apoenolase with magnesium, the metal that produces the highest level of activity, nickel, which produces a very low level, and calcium, which produces no activity, suggest strong binding of 2 mol (1 per subunit) of all three metals at the same sites, called “conformational” sites. About two-thirds of the possible absorbance change in the chromophoric competitive inhibitor 3-aminoenolpyruvate-2-phosphate (AEP) that occurs when it binds to the enzyme in the presence of saturating levels of magnesium is produced when just 2 mol (1 per subunit) of magnesium is added. Since additional “catalytic” metal won't bind unless the AEP does, and the AEP won't bind unless the “conformational” sites are filled with metal, much of the absorbance change in the AEP must be produced by conformational metal.Metals that do not produce enzymatic activity do not produce the absorbance change in AEP whereas metals that permit any level of enzymatic activity produce the same absorbance change that magnesium does-the reaction is “all or none.” Studies of the effect of calcium, nickel, and magnesium on the CD spectrum of apoenzyme-AEP solutions suggest that activating metals produce an asymmetric chromophore in the AEP. This is interprested as indicating the chromophore in AEP bound to enzyme in the presence of an activating metal is a twisted carbon-carbon double bond.Calorimetric studies show the competitive inhibitor 3-phosphoglycerate binds to the calcium- and magnesium-enzyme with about the same change in enthalpy. The substrate or AEP reduces the rate of the apparent reaction of the calcium- or magnesium-enzyme with excess EDTA, suggesting that both substrate and AEP bind to the calcium-enzyme. The interpretation of these data is that the conformational metal plays a crucial role in activating the substrate while the catalytic metal controls the reaction rate. This interpretation is supported by experiments in which an enzyme with one type of conformational metal is reacted in the stopped-flow with catalytic metal and substrate. If an activating metal is the conformational metal, the initial activity is greater.     

Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening

The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ). Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent. Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C). The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C. Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C. The corresponding delta Cp degrees value is approximately -2.4 kcal/deg. We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Exploring the energy landscape for Q(A)(-) to Q(B) electron transfer in bacterial photosynthetic reaction centers: effect of substrate position and tail length on the conformational gating step

The ability to initiate reactions with a flash of light and to monitor reactions over a wide temperature range allows detailed analysis of reaction mechanisms in photosynthetic reaction centers (RCs) of purple bacteria. In this protein, the electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) is rate-limited by conformational changes rather than electron tunneling. Q(B) movement from a distal to a proximal site has been proposed to be the rate-limiting change. The importance of quinone motion was examined by shortening the Q(B) tail from 50 to 5 carbons. No change in rate was found from 100 to 300 K. The temperature dependence of the rate was also measured in three L209 proline mutants. Under conditions where Q(B) is in the distal site in wild-type RCs, it is trapped in the proximal site in the Tyr L209 mutant [Kuglstatter, A., et al. (2001) Biochemistry 40, 4253-4260]. The electron transfer slows at low temperature for all three mutants as it does in wild-type protein, indicating that conformational changes still limit the reaction rate. Thus, Q(B) movement is unlikely to be the sole, rate-limiting conformational gating step. The temperature dependence of the reaction in the L209 mutants differs somewhat from wild-type RCs. Entropy-enthalpy compensation reduces the difference in rates and free energy changes at room temperature.     

A model for calculating the Bohr effect in hemoglobin equilibria.

The unusual aspects of the reaction of oxygen with hemoglobin are believed to be due to the free energy of the conformational change in the hemoglobin molecule upon oxygenation. The conformational free energy change due to oxygenation can be estimated in terms of the surface free energy of an emuslion droplet of the same size as the hemoglobin molecule. Calculations on the basis of this model lead to an equilibrium constant that varies with pH as in the acid and alkaline Bohr Effects, and that also varies with the ionic strength. The model used in this paper provides a simple way of estimating the variation of the equilibrium constant of a reaction involving a globular protein where the free energy of conformational changes can be evaluated in terms of surface properties.     

Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation

The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Impairment of the catalytic activity of Escherichia coli ribonucleic acid polymerase by a unique reaction of 4-chloro-7-nitrobenzofurazan.

Escherichia coli RNA polymerase loses 55-65% of its catalytic activity on reaction with Nbf-Cl (4-choro-7-nitrobenzofurazan). This partial inactivation was shown to be the result of specific impairment of RNA-chain elongation, since initiation of RNA chains was not altered after treatment with Nbf-Cl. The site of reaction was shown to be a unique thiol on the beta-subunit. This thiol is not accessible to reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). No protection of the enzyme against reaction with Nbf-Cl could be obtained with the inhibitor rifamycin nor with calf thymus DNA, GTP or 1,10-phenanthroline, indicating that the unique thiol is probably not within the active site. The specific impairment of RNA-chain elongation thus appears to be the result of a local conformational change which leaves chain initiation unimpaired. Changes observed in the tryptophan fluorescence spectrum of the enzyme or reaction with Nbf-Cl are consistent with formation of a Meisenheimer complex of the reagent with a nucleophilic group on the enzyme near the reactive thiol. It is proposed that formation of such a complex and a subsequent conformational change renders this thiol unusually susceptible to reaction with Nbf-Cl.     

Quenching of protein fluorescence by transient intermediates in the liver alcohol dehydrogenase reaction.

The addition of saturating concentrations of NAD-+ and alcohol to liver alcohol dehydrogenase in a stopped flow fluorimeter results in a triphasic quenching of enzyme fluorescence. A rapid quenching occurs with a rate constant of 300 to 500 s-minus 1, followed by a slower reaction at 50 to 100 s-minus 1, and ultimately followed by a very slow reaction. The addition of NAD-+ to enzyme in the absence of substrate causes a rapid quenching of enzyme fluorescence at 300 to 500 s-minus 1, with the same amplitude as the rapid phase in the presence of substrate. These studies demonstrate that NAD-+ binding to liver alcohol dehydrogenase causes a conformational change at a rate compatible with the previously reported rate constant for proton release, indicating that proton release is probably coupled to the conformational change.     

Streptolysin O: inhibition of the conformational change during membrane binding of the monomer prevents oligomerization and pore formation

Streptolysin O is a four-domain protein toxin that permeabilizes animal cell membranes. The toxin first binds as a monomer to membrane cholesterol and subsequently assembles into oligomeric transmembrane pores. Binding is mediated by a C-terminally located tryptophan-rich motif. In a previous study, conformational effects of membrane binding were characterized by introducing single mutant cysteine residues that were then thiol-specifically derivatized with the environmentally sensitive fluorophoracrylodan. Membrane binding of the labeled proteins was accompanied by spectral shifts of the probe fluorescence, suggesting that the toxin molecule had undergone a conformational change. Here we provide evidence that this change corresponds to an allosteric transition of the toxin monomer that is required for the subsequent oligomerization and pore formation. The conformational change is reversible with reversal of binding, and it is related to temperature in a fashion that closely parallels the temperature-dependency of oligomerization. Furthermore, we describe a point mutation (N402E) that, while compatible with membrane binding, abrogates the accompanying conformational change. At the same time, the N402E mutation also abolishes oligomerization. These findings corroborate the contention that the target membrane acts as an allosteric effector to activate the oligomerizing and pore-forming capability of streptolysin O.     

Kinetics of the conformational change of skeletal troponin C induced by Ca2+-Mg2+ exchange at the high-affinity Ca2+-binding sites

The rate constant of the conformational change of skeletal troponin C (TnC) induced by the Ca2+ binding reaction with the high-affinity Ca2+-binding sites was determined in the presence of Mg2+ by the fluorescence stopped-flow method in 0.1 M KCl, 50 mM Na-cacodylate-HCl pH 7.0 at 20 degrees C. The [MgCl2] dependence of the rate constants of the observed biphasic conformational change leveled off at the high [MgCl2] region: the rate constants were 60 +/- 9 s-1 and 8 +/- 2 s-1, respectively. These values are larger than the rate constants of the biphasic fluorescence intensity change of TnC induced by Mg2+ removal reaction at the high-affinity Ca2+-binding sites (37 +/- 7 s-1 and 3.0 +/- 0.6 s-1) under the same experimental conditions. These results suggest that the Ca2+-Mg2+ exchange reaction at the high-affinity Ca2+-binding sites is faster than the resultant conformational change accompanying the fluorescence intensity change. Based on these results, we also reexamine the molecular kinetic mechanism of the conformational change of the protein induced by the Mg2+ binding or removal reaction with the high affinity Ca2+-binding sites of skeletal TnC.     

Photoactivation and conformational gating for manganese binding and oxidation in bacterial reaction centers

The influence of illumination history of native bacterial reaction centers (BRCs) on the ability of binding and photo-induced oxidation of manganous ions was investigated in the pH range between 8.0 and 9.4. Binding of manganous ions to a buried site required 6 to 11-fold longer incubation periods, depending on the pH, in dark-adapted BRCs than in BRCs that were previously illuminated prior to manganese binding. The intrinsic electron transfer from the bound manganese ion to the photo-oxidized primary electron donor was found to be limited by a 2 to 5-fold slower precursor conformational step in the dark-adapted samples for the same pH range. The conformational gating could be eliminated by photoactivation, namely if the BRCs were illuminated prior to binding. Unlike in Photosystem II, photoactivation in BRCs did not involve cluster assembly. Photoactivation with manganese already bound was only possible at elevated detergent concentration. In addition, also exclusively in dark-adapted BRCs, a marked breaking point in the Arrhenius-plot was discovered around 15 °C at pH 9.4 indicating a change in the reaction mechanism, most likely caused by the change of orientation of the 2-acetyl group of the inactive bacteriochlorophyll monomer located near the manganese binding site.     

Conformational change of dihydrofolate reductase near the active site after thiol modification: detected by limited proteolysis

Kinetic studies of chicken liver dihydrofolate reductase (CL-DHFR) and Chinese hamster ovary DHFR (CH-DHFR) activated following p-hydroxymercuribenzoate (p-HMB) modification indicate a conformational change at the active site, suggesting a loosening of the enzyme structure upon SH modification. In the present study, limited proteolysis was applied to detect the subtle conformational changes in SH-modified DHFRs. The digested peptide fragments were separated by Tricine SDS-PAGE and sequenced by Edman auto-degradation. The thiol modifier N-iodoacetyl-N'-(5-sulfo-1-nophthyl) ethylenediamine (IAEANS), which activates these DHFRs only weakly, was used as a control. The results of sequencing showed that compared to native enzyme, there is one additional cleavage site near the active site in p-HMB-modified CL-DHFR, two additional sites in p-HMB-modified CH-DHFR, but no additional site for IAEANS-modified DHFRs. These results indicate that activation of DHFRs following thiol modification is accompanied by a conformational change at or near the active site. This subtle change in the active site conformation results in a pronounced change in enzyme activity. This provides further evidence that flexibility at the active site is essential for full expression of enzyme catalytic activity. Comparing results obtained from previous experiments on guanidine- and urea-activated CL-DHFR, this shows that a conformational change near helix(28-39) is sufficient for full activation of DHFR.     

Conformations et spectre Raman de l'acide ribonucléique de transfert, tRNA

The structure of yeast tRNA^Asp in aqueous solution has been studied in sight of Raman spectra recorded between 5 and 82°C. A conformational change is evidenced at 20°C and an endomelting is found around 70°C. This melting temperature, much higher than in tRNA-^Phe (near 50°C) is interpreted by the presence of a higher number of G-C bases in tRNA^Asp.At a same temperature, the Raman spectrum of a tRNA^Asp crystal is quasi-identical than that of an aqueous solution, indicating a high structural similarity except bands corresponding to G,C bases which show a more effective stacking of these bases in the solid.     

Crystal structure of argininosuccinate synthetase from Thermus thermophilus HB8. Structural basis for the catalytic action

Argininosuccinate synthetase catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate. The three-dimensional structures of the enzyme from Thermus thermophilus HB8 in its free form, complexed with intact ATP, and complexed with an ATP analogue (adenylyl imidodiphosphate) and substrate analogues (arginine and succinate) have been determined at 2.3-, 2.3-, and 1.95-A resolution, respectively. The structure is essentially the same as that of the Escherichia coli argininosuccinate synthetase. The small domain has the same fold as that of a new family of "N-type" ATP pyrophosphatases with the P-loop specific for the pyrophosphate of ATP. However, the enzyme shows the P-loop specific for the gamma-phosphate of ATP. The structure of the complex form is quite similar to that of the native one, indicating that no conformational change occurs upon the binding of ATP and the substrate analogues. ATP and the substrate analogues are bound to the active site with their reaction sites close to one another and located in a geometrical orientation favorable to the catalytic action. The reaction mechanism so far proposed seems to be consistent with the locations of ATP and the substrate analogues. The reaction may proceed without the large conformational change of the enzyme proposed for the catalytic process.     

A temperature dependent transition in the Pribnow box of the trp promoter

Proton NMR spectra of the trp operator-promoter (sequence CGTACTAGTT.AACTAGTACG) show selective changes in chemical shift and relaxation rates over the range of temperature 0-45 degrees C for the non-exchangeable protons of A11 and A12 only. These bases are in the centre of the Pribnow box. The changes imply that at least three conformational states become significantly populated in this range of temperature, and probably involve a change in the propellor twists of A11 and A12 for one transition, and changes in the helical twist and local pitch for the other. As (1) mutations in the Pribnow box that destroy the TAA sequence impair promoter activity, and (2) the abortive initiation assay for RNA polymerase shows a transition near 20 degrees C, we propose that the observed conformational transitions in the trp promoter are an essential feature of good promoters.