The kinetics of a two-state transition of myosin subfragment 1. A temperature-jump relaxation study.

Temperature-jump measurements were carried out on myosin subfragment 1 (S1) labeled at Cys-707 with 5-(iodoacetamido)fluorescein (S1-AF). The relaxation was monitored by following the increase in the fluorescence intensity of the attached probe after a jump of 5.8 degrees C. A single relaxation process was observed over a range of final temperatures, and the relaxation time decreased from 16.69 ms at 15 degrees C to 3.91 ms at 27 degrees C. The relaxation results are interpreted in terms of a two-state transition: (S1-AF)L K+ in equilibrium with K- (S1-AF)H, and the observed single relaxation time (tau) equals l/(k(+) + k-). The individual first-order rate constants, k+ and k-, were calculated from tau and the equilibrium constant previously determined. The activation energy was 21.9 kcal/mol for the forward reaction and 9.3 kcal/mol for the reverse reaction, corresponding to an enthalpy value of 12.6 kcal/mol for the two-state transition. The results provide, for the first time, direct kinetic evidence of a two-state transition of S1 in the absence of bound nucleotide, and support a two-state model of unliganded myosin subfragment 1.     

Pressure-jump study of the kinetics of ethidium bromide binding to DNA

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and dynamics of a tryptophanepeptide-polynucleotide complex

The binding of LysTrpLys to single stranded poly(A) was studied by measurements of fluorescence, UV-absorbance, electrodichroism and field jump relaxation. The van't Hoff enthalpy determined at constant degree of peptide protonation is -3.5 kcal/mol (delta S = 9 e.u.). The electrodichroism of bound tryptophane residues is negative; its absolute value decreases with increasing degree of binding theta. The magnitude of the dichroism at low theta indicates a preferential orientation of the tryptophane residues in the plane of the adenine bases, suggesting stacking of Trp with adenine bases. The overall degree of orientation decreases, however, to virtually zero at high theta. Relaxation measurements by low theta demonstrate the existence of two steps in the binding reaction of LysTrpLys to poly(A): a fast bimolecular step controlled by diffusion is followed by a slow intramolecular conversion with a forward rate of 1.5 x 10(5) s-1 and a backward rate of 2.7 x 10(3) s-1. The forward rate is close to that expected for an insertion reaction into stacked poly(A), yet the corresponding stability constant (approximately 55) is unexpectedly high.     

Frequency domain measurements of the fluorescence lifetime of ribonuclease T1.

Using multifrequency phase/modulation fluorometry, we have studied the fluorescence decay of the single tryptophan residue of ribonuclease T1 (RNase T1). At neutral pH (7.4) we find that the decay is a double exponential (tau 1 = 3.74 ns, tau 2 = 1.06 ns, f1 = 0.945), in agreement with results from pulsed fluorometry. At pH 5.5 the decay is well described by a single decay time (tau = 3.8 ns). Alternatively, we have fitted the frequency domain data by a distribution of lifetimes. Temperature dependence studies were performed. If analyzed via a double exponential model, the activation energy for the inverse of the short lifetime component (at pH 7.4) is found to be 3.6 kcal/mol, as compared with a value of 1.0 kcal/mol for the activation energy of the inverse of the long lifetime component. If analyzed via the distribution model, the width of the distribution is found to increase at higher temperature. We have also repeated, using lifetime measurements, the temperature dependence of the acrylamide quenching of the fluorescence of RNase T1 at pH 5.5. We find an activation energy of 8 kcal/mol for acrylamide quenching, in agreement with our earlier report.     

Chemical modification and fluorescence labeling study of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum using iodoacetamide and its N-substituted derivatives

Sarcoplasmic reticulum membrane vesicles from rabbit skeletal muscle were treated with iodoacetamide (IAA) at pH 7.0 and 30 degrees C. At 1.0 mM IAA, 1 mol of IAA per mol of ATPase peptide was bound in 1 h. Under these conditions, IAA was attached specifically to the B-tryptic fragment portion of the peptide. The binding of IAA did not affect the Ca2+-transporting activity of ATPase. Three fluorescent derivatives of iodoacetamide, 5-(2-acetamidoethyl)aminonaphthalene-1-sulfonate (IAEDANS), 5-iodoacetamido fluorescein (IAF), and 5-iodoacetamido eosin (IAE), were also tested for reactivity toward sarcoplasmic reticulum ATPase at 30 degrees C and pH 7.0. In 1 h at 50 microM concentration, each of these fluorescent labels modified ATPase to a labeling density of 1 mol per mol of ATPase. Neither IAEDANS nor IAF at this labeling density affected Ca2+-transporting activity, but IAE reduced it to 20% of the untreated control. The target site of IAEDANS at this labeling density was located exclusively on the B-fragment portion, as was the case with IAA, but IAF label was found on both A1 and B fragments after limited tryptic digestion. IAEDANS was used as a B-fragment portion-directed conformational probe of Ca2+-transport ATPase, and an increase in fluorescence intensity accompanying E1Ca-P formation was detected. The fluorescence enhancement was abolished when E1Ca-P X ADP beta S was formed by adding ADP beta S to preformed E1Ca-P. This suggests that the conformation of ATPase in the neighborhood of the IAEDANS binding site may be altered in response to the dissociation of ADP from the phosphorylated intermediate.     

Helix–coil transition kinetics in aqueous poly(α,L-glutamic acid)

A polarimetric electric-field-jump relaxation apparatus is described and used to determine the relaxation spectrum for the helix–coil transition of poly(α,L -glutamic acid) in water at 24°C. A maximum relaxation time of 1.7 μc occurs at the transition midpoint (pH = 5.9) yielding a rate constant for helical growth of 6 × 10⁷ sec^?1.     

Photoconformational relaxation of protein structure from the data of tryptophan fluorescence

It was shown that the decrease of polarization degree with increasing the wavelength of protein tryptophane fluorescence lambda f was caused by the lengthening of the lifetime tau. A considerable rotational nanosecond mobility of the internal tryptophanyls was observed. This mobility is not spontaneous but caused by photoexcitation. The increase of tau with lambda f is observed not only for multitryptophan proteins but also for monotryptophan ones and for DL-tryptophane in glycerol. The increase of tau is caused by relaxation processes occurring in close proximity to tryptophane after its photoexcitation and reflects the photoinduced nanosecond mobility of the tryptophane microenvironment. Photoconformational relaxation of protein in the nanosecond time range and possible role of this process in photoinactivation of enzymes are discussed.     

Effects of the anesthetic dibucaine on the kinetics of the gel-liquid crystalline transition of dipalmitoylphosphatidylcholine multilamellar vesicles.

The effects of the anesthetic dibucaine on the relaxation kinetics of the gel-liquid crystalline transition of dipalmitoylphosphatidylcholine (DC16PC) multilamellar vesicles have been investigated using volume-perturbation calorimetry. The temperature and pressure responses to a periodic volume perturbation were measured in real time. Data collected in the time domain were subsequently converted into and analyzed in the frequency domain using Fourier series representations of the perturbation and response functions. The Laplace transform of the classical Kolmogorov-Avrami kinetic relation was employed to describe the relaxation dynamics in the frequency domain. The relaxation time of anesthetic-lipid mixtures, as a function of the fractional degree of melting, appears to be qualitatively similar to that of pure lipid systems, with a pronounced maximum, tau max, observed at a temperature corresponding to greater than 75% melting. The tau max decreases by a factor of approximately 2 as the nominal anesthetic/lipid mole ratio increases from 0 to 0.013 and exhibits no further change as the nominal anesthetic/lipid mole ratio is increased. However, the fractional dimensionality of the relaxation process decreases monotonically from slightly less than two to approximately one as the anesthetic/lipid mole ratio increases from 0 to 0.027. At higher ratios, the dimensionality appears to be less than one. These results are interpreted in terms of the classical kinetic theory and related to those obtained from Monte Carlo simulations. Specifically, low concentrations of dibucaine appear to reduce the average cluster size and cause the fluctuating lipid clusters to become more ramified. At the highest concentration of dibucaine, where n < 1, the system must be kinetically heterogeneous.     

Fractional order viscoelasticity of the aortic valve cusp: an alternative to quasilinear viscoelasticity

BACKGROUND: Quasilinear viscoelasticity (QLV) theory has been widely and successfully used to describe the time-dependent response of connective tissues. Difficulties remain, however, particularly in material parameter estimation and sensitivities. In this study, we introduce a new alternative: the fractional order viscoelasticity (FOV) theory, which uses a fractional order integral to describe the relaxation response. FOV implies a fractal-like tissue structure, reflecting the hierarchical arrangement of collagenous tissues. METHOD OF APPROACH: A one-dimensional (I-D) FOV reduced relaxation function was developed, replacing the QLV "box-spectrum" function with a fractional relaxation function. A direct-fit, global optimization method was used to estimate material parameters from stress relaxation tests on aortic valve tissue. RESULTS: We found that for the aortic heart valve, FOV had similar accuracy and better parameter sensitivity than QLV, particularly for the long time constant (tau2). The mean (n = 5) fractional order was 0.29, indicating that the viscoelastic response of the tissue was strongly fractal-like. RESULTS SUMMARY: mean QLV parameters were C = 0.079, tau1 = 0.004, tau2 = 76, and mean FOV parameters were beta = 0.29, tau = 0.076, and rho = 1.84. CONCLUSIONS: FOV can provide valuable new insights into tissue viscoelastic behavior Determining the fractional order can provide a new and sensitive quantitative measure for tissue comparison.     

Inactivation of fructose-1,6-bisphosphatase by o-phthalaldehyde

Rabbit liver fructose-1,6-bisphosphatase, a tetramer of identical subunits was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The second-order rate constant for the inactivation was 30 M-1s-1. Fructose-1,6-bisphosphatase was completely protected from inactivation by the substrate--fructose-1,6-diphosphate but not by the allosteric effector--adenosine monophosphate. The absorption spectrum (lambda max 337 nm) and, fluorescence excitation (lambda max 360 nm) and fluorescence emission spectra (lambda max 405 nm) were consistent with the formation of an isoindole derivative in the subunit between a cysteine and a lysine residue about 3A apart. About 4 isoindole groups per mol of the bisphosphatase were formed following complete loss of the phosphatase activity. This suggests that the amino acid residues of the biphosphatase participating in reaction with o-phthalaldehyde more likely reside at or near the active site instead of allosteric site. The molar transition energy of fructose-1,6-bisphosphatase--o-phthalaldehyde adduct was estimated 121 kJ/mol and compares favorably with 127 kJ/mol for the synthetic isoindole, 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl) isoindole in hexane. It is, thus, concluded that the cysteine and lysine residues participating in isoindole formation in reaction between fructose-1,6-bisphosphatase and o-phthalaldehyde are located in a hydrophobic environment.     

Segmental flexibility of single-, double-, and triple-stranded polyribonucleotides from the data of spin label method. Formation of the triple-stranded poly(A.A.U) polynucleotide helix

Segmental mobility dynamic peculiarities of poly(U), poly(A) and poly(C) synthetic polymers and their complexes were investigated by spin-label method. Imidazolide spin-label was introduced into 2'-oxi-groups of polymer ribose in correlation: one spin-label on 18-20 bases. Formation of complexes was observed by ESR spectra at two pH: 4.2 and 7.2. Segmental mobility of only single strand spin-labelled polymer segment and in the complex was evaluated by measuring rotational correlation time (tau) determined by dependence of distances between outer wide extrema in ESR spectra from solvent viscosity at different temperatures. It turned out that correlation time tau of single strand structures in a high degree depend on pH and temperature. For three strand structures abrupt increase of tau because of appearance of rigidity was observed. It is possible to evaluate part of triple complexes poly(U.A.A) and poly(U.U.A) existing in dynamic equilibrium depending on pH and temperature by the form of outer wide extrema. Adding of dye to complex of poly(U).poly(A) causes an increase of rigidity of the supermolecular structure. Quantitative characteristics of formed complexes were obtained by simulation of ESR spectra on computer.     

Dynamic equilibrium between the two conformational states of spin-labeled tropomyosin

Tropomyosin was labeled with a maleimide nitroxide spin-label attached to cysteine-190 via a succinimido ring which was subsequently opened by incubation at alkaline pH. Electron spin resonance (ESR) spectra showed a temperature-dependent equilibrium, below the main unfolding transition of tropomyosin, between labels which were restricted in their motion (strongly immobilized), predominating at low temperatures, and those which were highly mobile (weakly immobilized), predominating at higher temperatures. These label states were associated with two protein states from a comparison of the ESR spectral changes with the thermal unfolding profile of tropomyosin. The strongly immobilized labels were associated with the completely folded molded and the weakly immobilized labels with a partially unfolded (in the cysteine-190 region) state which is an intermediate in the thermal unfolding of tropomyosin. A spectral subtraction technique was used to measure the concentration ratio of strongly and weakly immobilized labels from which an equilibrium constant, K, was determined at different temperatures. A linear van't Hoff plot was obtained, indicating that the spin-labeled protein is in thermal equilibrium between these two conformational states with delta H = 17 kcal/mol, delta S = 56 cal/(deg X mol), and K = 1.0 at 34 degrees C. An upper limit of 10(7) s-1 for the conformational fluctuation was estimated from the shapes and separation of the two ESR spectral components. In contrast to the label with the opened succinimido ring, the spin-label with an intact succinimido ring remained strongly immobilized on the protein, indicating that in the partially unfolded state the molecule retains structure in the cysteine-190 region.     

Spin labeled nucleic acids.

Homopolyribonucleotides and E. coli DNA wer spin labeled with an iodoacetamide-nitroxide compound. The extent of labeling is highly dependent upon the nature of the base and the secondary structure of the nucleic acid. This spin label-polymer linkage is unstable at high temperatures and in phosphate buffers. In order to determine the effect of changes in the environment of nucleic acids on the esr signals of their attached spin labels, the polynucleotides were subjected to temperature and viscosity perturbations. An increase in temperature (T) affects a linear decrease in the anisotropy factor of the esr signal. The log tau (tau = correlation time) versus (1/T) profile is linear with a positive slope when the spin label is attached to single stranded polynucleotides but exhibits discontinuities at certain critical temperatures when attached to the duplexes poly (As-U) and poly (I-Cs). These critical temperatures are lower than the optical Tm. Logarithmic increase in viscosity was found to produce a linear increase in tau in aqueous sucrose solutions.     

Influence of various fluorescent and non-fluorescent labels on the kinetics of the complex formation of alpha-chymotrypsin with basic pancreatic trypsin inhibitor (Kunitz).

The association of alpha-chymotrypsin with basic pancreatic trypsin inhibitor was studied using extrinsic signals produced by fluorescent and nonfluorescent labels. The reactive dyes were covalently bound to the proteins in the complexed state, in which the binding region was protected. The signals were sufficiently large to measure the complex formation at protein concentrations of 10(-9)M by fluorescence and down to 10(-6)M by absorption. Therefore, the association and dissociation could be followed over a broad range of concentration. Good correspondence was observed between data which were obtained with different labels and with published values for the unlabeled proteins. Existing differences could be explained by different buffer conditions used by the different authors. Also the pH dependence of the dissociation rate constants was essentially unaltered by the introduction of the labels. The large signals allowed a direct measurement of the equilibrium constants of dissociation, even at high pH, at which they are in the range of 10(-8)M. The experimentally determined binding constants were in agreement with those calculated from the rate constants. The temperature dependence of the binding constants revealed a small positive and pH-dependent enthalpy change [deltaHo = 4.0 kcal/mol (16.7 kJ) at H 7.0[. The results prove that the labeling can be performed in such a way that the equilibrium and kinetic parameters of the system studied are not significantly influenced.     

Effect of cations on the quantum yield and the lifetime of the chloroplast fluorescence]

The interrelationship between the cation-induced fluorescence changes and the state of the photosystem 2 (PS-2) reaction centers for pea chloroplasts and their osmotic fragments was studied. The effects of K+ and Mg2+ on the fluorescence quantum yield (phi f1) under varying light intensities as well as on the fluorescence lifetime (tau f1) in the saturating light were demonstrated. K+ induces the decrease in tau f1; Mg2+ exerts an opposite effect. The effects were more pronounced when the reaction centers of PS-2 were converted into an inactive state by illuminating the sample with a saturating light or by adding DCMU. Under these conditions the cations' effect on tau f1 was accompanied by proportional changes in tau f1. It was concluded that in Mg-deficient chloroplasts an efficient channel of the excitation quenching appears in antenna chlorophyll of PS-2 with the rate constant of 7 . 10(8) s-1. The simultaneous measurements of tau f1 by phase and modulation type techniques allowed to reveal the emission heterogeneity within the nanosecond time interval and the DCMU-sensitive delayed fluorescence with the lifetime exceeding 10(-7) s and the overall quantum yield approximately equal to 2 . 10(-3).     

Structural features of the binding site of cholera toxin inferred from fluorescence measurements

The dependence on pH of the fluorescence of cholera toxin and its A and B subunits has been studied at 25 degrees C. The fluorescence intensity of cholera toxin is highly pH-dependent. In the pH range 7-9.5 it reaches a maximum corresponding to a quantum yield of 0.076. In the pH range 4-7 a strong increase in fluorescence intensity is observed (delta Q/Qmax = 0.64). Evaluation of the pH sensitivity of the fluorescence intensity of the A and B subunits reveals that the B subunit is mainly responsible for the observed pH effect (delta Q/Qmax for B subunit = 0.64). The intensity changes are paralleled by similar although less pronounced changes in the average fluorescence excited state life-time tau (delta tau/tau max = 0.33 for cholera toxin). Fluorimetric titration of the B subunit, which is related to the indole fluorescence of the lone Trp-88, reveals that the fluorescence intensity changes in the pH range 4-7 are due to reaction of two types of ionizable quencher displaying apparent pKa values of 4.4 and 6.2, respectively. It is suggested that the increase in fluorescence intensity with a midpoint at pH 6.2 is the result of deionization of the imidazolium side-chain of one or two out of the four histidine residues present in each beta-polypeptide chain, whereas a deionized carboxyl group is responsible for the quenching with midpoint at pH 4.4. Complex formation of cholera toxin or B subunit with the monosialoganglioside GM1 or the oligosaccharide moiety of GM1 (oligo-GM1) completely prevents the quenching by both quenchers. Addition of 6 M urea also eliminates the pH effect. The quenching is not the result of the dissociation of the B subunit into its constituent monomers. Upon fluorimetric titration of cholera toxin or B subunit above pH 9, a progressive drop in both fluorescence intensity and tau occurs. This decrease could be due to energy transfer from the indole moiety of Trp-88 to ionized tyrosines or by quenching through an unprotonated epsilon-amino group of lysine. Fluorimetric titration of the A subunit indicates that the tryptophan fluorescence is only moderately altered by ionizable groups displaying a pKa in the range 4 to 9. Activation of A subunit does not affect this lack of pH sensitivity. Above pH 9, however, a much more significant drop in the fluorescence intensity of activated A subunit occurs. The structural implications of the results are discussed.     

Homonyms, synonyms and mutations of the sequence/structure vocabulary

The effect of pH and temperature on the association equilibrium constant (Ka) for bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B has been investigated. Ka values decrease with decreasing pH, reflecting the acid-midpoint and pK shifts, upon BPTI binding, of a three-proton co-operative transition, between pH 3 and 5, and of a single ionizable group, between pH 5 and 9. At pH 8, the values of delta H degree (between 7 degrees C and 42 degrees C) and delta S degree (at 21 degrees C) for BPTI binding to the glandular kallikreins considered were determined. In particular, the delta H degree values have been found to be independent of temperature and the following values have been obtained by van't Hoff plots: +1.8 kcal/mol, +2.3 kcal/mol and +2.4 kcal/mol (1 kcal = 4184 J) for the inhibitor binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B, respectively. Considering the known molecular structures of free porcine pancreatic beta-kallikrein A and BPTI, and of their complex, the stereochemistry of the enzyme : inhibitor contact regions was analysed for the three serine proteinases, in relation to their respective types of behaviour.     

Pre-steady state kinetics of the ferroxidase reaction catalyzed by ceruloplasmin

Analysis of the presteady-state step of the ferroxidase reaction catalyzed by ceruloplasmin revealed that the kinetic curve for the reaction product accumulation versus time is characterized by an induction period, tau, whose value does not change within the pH range of 5.0-7.5. The value of tau decreases with a rise in the enzyme concentration and increases with a rise in the substrate concentration at constant Fe(II) and E0 concentrations. A reaction scheme is proposed which points to the existence of an induction period in the ceruloplasmin-catalyzed ferroxidase reaction. It was shown that the calculated values of tau = f([Fe(II)]0) and tau = f([E]0) obtained through the use of differential equations qualitatively coincide with the corresponding experimental values.