Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC

Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C.     

A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies

Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.     

Recognition between disordered states: kinetics of the self-assembly of thioredoxin fragments

The disordered N- (1-73) and C- (74-108) fragments of oxidized Escherichia colithioredoxin (Trx) reconstitute the native structure upon association [Tasayco, M. L., & Chao, K. (1995) Proteins: Struct., Funct., Genet. 22, 41-44]. Kinetic measurements of the formation of the complex (1-73/74-108) at 20 degrees C under apparent pseudo-first-order conditions using stopped-flow far-UV CD and fluorescence spectroscopies indicate association coupled to folding, an apparent rate constant of association [kon = (1330 +/- 54) M-1 s-1], and two apparent unimolecular rate constants [k1 = (0. 037 +/- 0.007) s-1 and k2 = (0.0020 +/- 0.0005) s-1]. The refolding kinetics of the GuHCl denatured Trx shows the same two slowest rate constants. An excess of N- over C-fragment decreases the kon, and the slowest phase disappears when a P76A variant is used. Stopped-flow fluorescence measurements at 20 degrees C indicate a GuHCl-dependent biphasic dissociation/unfolding process of the complex, where the slowest phase corresponds to 90% of the total. Their rate constants, extrapolated to zero denaturant, k-1 = (9 +/- 3) x 10(-5) s-1 and k-2 = (3.4 +/- 1.2) x 10(-5) s-1, show m# values of (4.0 +/- 0.4) kcal mol-1 M-1 and (3.5 +/- 0.1) kcal mol-1 M-1, respectively. Our results indicate that: (i) a compact intermediate with trans P76 and defined tertiary structure seems to participate in both the folding and unfolding processes; (ii) not all the N-fragment is competent to associate with the C-fragment; (iii) conversion to an association competent form occurs apparently on the time scale of P76 isomerization; and (iv) the P76A variation does not alter the association competency of the C-fragment, but it permits its association with "noncompetent" forms of the N-fragment.     

Unfolding of the tetrameric loop deletion mutant of ROP protein is a second-order reaction

Comprehensive kinetic studies were carried out on the unfolding properties of RM6 as a function of GdnHCl concentration and temperature. This protein is a mutant resulting from the dimeric wild-type CoLE1-ROP protein by deletion of 5 amino acids (Asp 30, Ala 31, Asp 32, Glu 33, Gln 34) in the loop of each monomer. The deletion has dramatic consequences. The dimeric 4-alpha-helix structure characteristic of the wild-type protein is completely reorganized and the RM6 structure can be described as a tetrameric alpha helix of extended monomers without loops. These extraordinary structural changes are accompanied by an enormous increase in transition temperature from 71 to 101 degreesC. These features have been discussed in a separate publication (1). The remarkable change in thermal stability of RM6 should be reflected in significant changes in the folding rate constants. This was observed in the present unfolding studies. Decay of tetrameric RM6 was monitored by circular dichroism (CD) and fluorescence to probe for changes in both secondary and tertiary structure, respectively. The identity of the kinetic parameters obtained from the two techniques supports the view that secondary and tertiary structure break down simultaneously. However, the most intriguing result is the finding that unfolding of tetrameric RM6 can be described very well by a second-order reaction. The magnitude of the second-order rate constant k2 varies dramatically with both temperature and denaturant concentration. At 25 degreesC and 6.5 M GdnHCl concentration k2 is 4200 L.(mol of dimer)-1.s-1, whereas at 4.4 M GdnHCl a value of k2 = 0.9 L.(mol of dimer)-1.s-1 is observed. Correspondingly, apparent activation enthalpies show a strong increase from DeltaH# = 29.1 kJ.mol-1 at 6. 5 M GdnHCl to Delta H# = 79.7 kJ.mol-1 at 4.4 M GdnHCl. A mechanism involving a dimeric intermediate is suggested which permits a consistent interpretation of the findings.     

Kinetics of binding of methyl alpha- and beta-D-galactopyranoside to peanut agglutinin: a carbon-13 nuclear magnetic resonance study

The binding kinetics of methyl alpha- and methyl beta-D-galactopyranoside to the anti-T lectin from peanuts were studied by 13C NMR, employing methyl galactopyranosides specifically enriched in 13C at C-1. Association and dissociation rate constants, as well as their activation parameters, are reported. The association rate constants, 4.6 X 10(4) M-1 s-1 for the alpha-galactopyranoside and 3.6 X 10(4) M-1 s-1 for the beta-galactopyranoside, are several orders of magnitude below those expected for a diffusion-controlled process. For both anomers, the association rate constant was temperature independent, implying that the association process occurs without a significant activation enthalpy. However, a considerable association activation entropy was found for both ligands. The dissociation rate constants were in the range of 9-46 s-1 within a temperature range of 5-35 degrees C for the alpha-galactopyranoside, and in the range of 9-39 s-1 within a temperature range of 5-25 degrees C for the beta-galactopyranoside. A considerable dissociation activation enthalpy of ca. 10 kcal mol-1 was found for both anomers. A two-step binding model, consistent with the present NMR data and with previous UV and CD spectroscopic data, is presented.     

Kinetic determination of the fast exchanging substrate water molecule in the S3 state of photosystem II

In a previous communication we showed from rapid isotopic exchange measurements that the exchangeability of the substrate water at the water oxidation catalytic site in the S3 state undergoes biphasic kinetics although the fast phase could not be fully resolved at that time [Messinger, J., Badger, M., and Wydrzynski, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3209-3213]. We have since improved the time resolution for these measurements by a further factor of 3 and report here the first detailed kinetics for the fast phase of exchange. First-order exchange kinetics were determined from mass spectrometric measurements of photogenerated O2 as a function of time after injection of H218O into spinach thylakoid samples preset in the S3 state at 10 degreesC. For measurements made at m/e = 34 (i. e., for the mixed labeled 16,18O2 product), the two kinetic components are observed: a slow component with k1 = 2.2 +/- 0.1 s-1 (t1/2 approximately 315 ms) and a fast component with k2 = 38 +/- 4 s-1 (t1/2 approximately 18 ms). When the isotopic exchange is measured at m/e = 36 (i.e., for the double labeled 18,18O2 product), only the slow component (k1) is observed, clearly indicating that the substrate water undergoing slow isotopic exchange provides the rate-limiting step in the formation of the double labeled 18,18O2 product. When the isotopic exchange is measured as a function of temperature, the two kinetic components reveal different temperature dependencies in which k1 increases by a factor of 10 over the range 0-20 degreesC while k2 increases by only a factor of 3. Assuming simple Arrhenius behavior, the activation energies are estimated to be 78 +/- 10 kJ mol-1 for the slow component and 39 +/- 5 kJ mol-1 for the fast component. The different kinetic components in the 18O isotopic exchange provide firm evidence that the two substrate water molecules undergo separate exchange processes at two different chemical sites in the S3 state, prior to the O2 release step (t1/2 approximately 1 ms at 20 degreesC). The results are discussed in terms of how the substrate water may be bound at two separate metal sites.     

Structure, interaction and electron transfer between cytochrome b5, its E44A and/or E56A mutants and cytochrome c

Site-directed mutagenesis has been used to produce variants of a tryptic fragment of bovine liver cytochrome b5 in which Glu44 and Glu56 are mutated to alanine. The reduction potentials measured by spectroelectrochemical titration (in the presence of 1 mM (Ru(NH3)6)3+, pH 7.0 and I=0.1 M) are 4.5, 6.0, 6.0 and 7.5 mV versus the standard hydrogen electrode (SHE) for the wild-type and E44A, E56A and E44/56A mutants of cytochrome b5, respectively. A comparative two-dimensional NMR study of cytochrome b5 and its E44/56A mutant in water solution has been achieved. Resonance assignments of side-chains have been completed successfully. The NMR results suggest that the secondary structures and global folding of the E44/56A mutant remain unchanged, but the mutation of both Glu44 and Glu56 to hydrophobic alanine may lead to the two helices containing mutated residues contracting towards the heme center. The inner mobility of the Gly42 approximately Glu44 segment in cytochrome b5 may be responsible for the difference of the binding mode between Glu44 and Glu56 with cytochrome c. The binding between cytochrome c and cytochrome b5 was studied by optical difference spectra of cytochrome c and variants of cytochrome b5. The association constants (KA) for the wild-type, E44A, E56A, and E44/56A mutants of cytochrome b5 with cytochrome c, are 4.70(+/-0. 10)x10(6) M-1, 1.88(+/-0.03)x10(6) M-1, 2.70(+/-0.13)x10(6) M-1, and 1.14(+/-0.05)x10(6) M-1, respectively. This is indicative that both Glu44 and Glu56 are involved in the complex formation between cytochrome b5 and cytochrome c. The reduction of horse heart ferricytochrome c by recombinant ferrocytochrome b5 and its mutants has been studied. The rate constant of the electron transfer reaction between ferricytochrome c and wild-type ferrocytochrome b5 (1.074(+/-0.49)x10(7) M-1 s-1) is higher than those of the mutant protein E44A (8.98(+/-0.20)x10(6) M-1 s-1), E56A (8.76(+/-0. 39)x10(6) M-1 s-1), and E44/56A (8.02(+/-0.38)x10(6) M-1 s-1) at 15 degreesC, pH 7.0, I=0.35 M. The rate constants are strongly dependent on ionic strength and temperature. These studies, by means of a series of techniques, provide conclusive results that the interaction between cytochrome b5 and cytochrome c is electrostatically guided, and, more importantly, that both Glu44 and Glu56 participate in the electron transfer reaction.     

Kinetic study of the reaction of glutathione peroxidase with peroxynitrite

Glutathione peroxidases and their mimics, e.g., ebselen or diaryl tellurides, efficiently reduce peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) to nitrite and protect against oxidation and nitration reactions. Here, we report the second-order rate constant for the reaction of the reduced form of glutathione peroxidase (GPx) with peroxynitrite as (8.0 +/- 0.8) x 10(6) M-1 s-1 (per GPx tetramer) at pH 7.4 and 25 degreesC. The rate constant for oxidized GPx is about 10 times lower, (0.7 +/- 0.2) x 10(6) M-1 s-1. On a selenium basis, the rate constant for reduced GPx is similar to that obtained previously for ebselen. The data support the conclusion that GPx can exhibit a biological function by acting as a peroxynitrite reductase.     

In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1. 34 x 10(4) M-1 s-1 ( approximately 25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2'-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 x 10(6) M-1 s-1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1 degreesC vs. 76.5 degreesC). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 x 10(-6) M-1 s-1 vs. 22.3 x 10(-6) M-1 s-1; koff, 24.3 s-1 vs. 24.2 s-1; KO2, 0.91 x 10(-6) M-1 vs. 0.92 x 10(-6) M-1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 x 10(-6) M-1s-1 vs. 0.51 x 10(-6) M-1s-1; koff, 5.08 s-1 vs. 3.51 s-1; KCO, 1.77 x 10(-7) M-1 vs. 1.45 x 10(-7) M-1). All four substitutions are in the heme pocket and within 5 A of the heme group.     

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus comprises an N-terminal domain (residues 1-319), which is dimeric and forms tyrosyladenylate, and a C-terminal domain (residues 320-419), which binds the anticodon arm of tRNATyr. The N-terminal domain has the characteristic fold of the class I aminoacyl-tRNA synthetases. The unfolding of the N-terminal domain by urea at 25 degreesC under equilibrium conditions was monitored by its intensities of light emission at 330 and 350 nm, the ratio of these intensities, its ellipticity at 229 nm, and its partition coefficient, in spectrofluorometry, circular dichroism, and size-exclusion chromatography experiments, respectively. These experiments showed the existence of an equilibrium between the native dimeric state of the N-terminal domain, a monomeric intermediate state, and the unfolded state. The intermediate was compact and had secondary structure, and its tryptophan residues were partially buried. These properties of the intermediate and its inability to bind 1-anilino-8-naphthalenesulfonate showed that it was not in a molten globular state. The variation of free energy deltaG(H2O) and its coefficient m of dependence on the concentration of urea were, respectively, 13.8 +/- 0.2 kcal.mol-1 and 0.9 +/- 0.1 kcal.mol-1.M-1 for the dissociation of the native dimer and 13.9 +/- 0.6 kcal.mol-1 and 2.5 +/- 0.1 kcal.mol-1.M-1 for the unfolding of the monomeric intermediate.     

Dimer-to-tetramer transformation: loop excision dramatically alters structure and stability of the ROP four alpha-helix bundle protein

The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the five amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-alpha-helix motif of the wild-type structure is given up. Instead a homotetrameric four-alpha-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71 degreesC characteristic of the wild-type protein to 101 degreesC for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer)-1 to 195.1 kJ (mol of tetramer)-1. The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of DeltaH=1073+/-30 kJ (mol of tetramer)-1 and a heat capacity change at the transition temperature of DeltaDNCp=14.9(+/-)3% kJ (mol of tetramerxK)-1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van't Hoff enthalpy values.     

Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure

A combination of circular dichroism spectroscopy, titration calorimetry, and optical melting has been used to investigate the association of the minor groove ligands netropsin and distamycin to the central A3T2 binding site of the DNA duplex d(CGCAAATTGGC).d(GCCAATTTGCG). For the complex with netropsin at 20 degrees C, a ligand/duplex stoichiometry of 1:1 was obtained with Kb approximately 4.3 x 10(7) M-1, delta Hb approximately -7.5 kcal mol-1, delta Sb approximately 9.3 cal K-1 mol-1, and delta Cp approximately 0. Previous NMR studies characterized the distamycin complex with A3T2 at saturation as a dimeric side-by-side complex. Consistent with this result, we found a ligand/duplex stoichiometry of 2:1. In the current study, the relative thermodynamic contributions of the two distamycin ligands in the formation of this side-by-side complex (2:1 Dst.A3T2) were evaluated and compared with the thermodynamic characteristics of netropsin binding. The association of the first distamycin molecule of the 2:1 Dst.A3T2 complex yielded the following thermodynamic profile: Kb approximately 3.1 x 10(7) M-1, delta Hb = -12.3 kcal mol-1, delta Sb = -8 cal K-1 mol-1, and delta Cp = -42 cal K-1 mol-1. The binding of the second distamycin molecule occurs with a lower Kb of approximately 3.3 x 10(6) M-1, a more favorable delta Hb of -18.8 kcal mol-1, a more unfavorable delta Sb of -34 cal K-1 mol-1, and a higher delta Cp of -196 cal K-1 mol-1. The latter term indicates an ordering of electrostricted and structural water molecules by the complexes. These results correlate well with the NMR titrations and are discussed in context of the solution structure of the 2:1 Dst.A3T2 complex.     

Kinetic properties of ba3 oxidase from Thermus thermophilus: effect of temperature

The kinetic properties of the ba3 oxidase from Thermus thermophilus were investigated by stopped-flow spectroscopy in the temperature range of 5-70 degrees C. Peculiar behavior in the reaction with physiological substrates and classical ligands (CO and CN-) was observed. In the O2 reaction, the decay of the F intermediate is significantly slower (k' = 100 s-1 at 5 degrees C) than in the mitochondrial enzyme, with an activation energy E of 10.1 +/- 0.9 kcal mol-1. The cyanide-inhibited ba3 oxidizes cyt c522 quickly (k approximately 5 x 10(6) M-1 s-1 at 25 degrees C) and selectively, with an activation energy E of 10.9 +/- 0.9 kcal mol-1, but slowly oxidizes ruthenium hexamine, a fast electron donor for the mitochondrial enzyme. Cyt c552 oxidase activity is enhanced up to 60 degrees C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c522-ba3 electrostatic complex. The thermophilic oxidase is less sensitive to cyanide inhibition, although cyanide binding under turnover is much quicker (seconds) than in the fully oxidized state (days). Finally, the affinity of reduced ba3 for CO at 20 degrees C (Keq = 1 x 10(5) M-1) was found to be smaller than that of beef heart aa3 (Keq = 4 x 10(6) M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a32+ of ba3 (k' = 0.8 s-1 vs k' = 0.02 s-1 for beef heart aa3 at 20 degrees C). The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O2 tensions is discussed.     

Transient kinetics of formation and reaction of the uridylyl-enzyme form of galactose-1-P uridylyltransferase and its Q168R-variant: insight into the molecular basis of galactosemia

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate (Gal-1-P) to form UDP-galactose and glucose 1-phosphate (Glc-1-P) through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme). Gln 168 in E. coli uridylyltransferase engages in hydrogen bonding with the phosphoryl oxygens of the UMP moiety, which is bonded to His 166 in the intermediate [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569]. In humans, the point variant Q188R accounts for 60% of galactosemia cases. The corresponding E. coli variant Q168R has been overexpressed and purified. In preparation for kinetic correlation of Q168R and wild-type uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme. At 4 degreesC, the first-order rate constant for uridylylation by UDP-glucose is 281 +/- 18 s-1, and for deuridylylation it is 226 +/- 10 s-1 with Glc-1-P and 166 +/- 10 s-1 with Gal-1-P. Inasmuch as the overall turnover number at 4 degreesC is 62 s-1, the covalent intermediate is kinetically competent. The variant Q168R is uridylylated by UDP-glucose to the extent of about 65% of the potential active sites. Uridylylation reactions of Q168R with UDP-glucose proceed with maximum first-order rate constants of 2.2 x 10(-)4 s-1 and 4.2 x 10(-)4 s-1 at 4 and 27 degreesC, respectively. In experiments with uridylyl-Q168R and glucose-1-P, the mutant enzyme undergoes deuridylylation with maximum first-order rate constants of 4.8 x 10(-)4 s-1 and 1.68 x 10(-)3 s-1 at 4 and 27 degreesC, respectively. The value of Km for uridylylation of Q168R is slightly higher than for the wild-type enzyme, and for deuridylylation it is similar to the wild-type value. The wild-type enzyme undergoes uridylylation and deuridylyation about 10(6) times faster than Q168R. The wild-type activity in the overall reaction is 1.8 x 10(6) times that of Q168R. The wild-type enzyme contains 1.9 mol of Zn+Fe per mole of subunits, whereas the Q168R-variant contains 1.36 mol of Zn+Fe per mole of subunits. The mutation stabilizes the uridylyl-enzyme by 1.2 kcal mol-1 in comparison to the wild-type enzyme. These results show that the low activity of Q168R is not due to overstabilization of the intermediate or to the absence of structural metal ions. Instead, the main defect is very slow uridylylation and deuridylation.     

Assessment of the degree of disability in narcolepsy

The effect of free fatty acids (FFA) and non-enzymatic glycation on the binding kinetics of dansylsarcosine (DS) to human serum albumin (HSA) was studied using the stopped-flow technique. The influence of FFA on the binding parameters of 25% glycated HSA depended on the type of fatty acid. The addition of stearic, oleic and linoleic acids in a concentration of 0.3 mmol/l showed no inhibitory effects on the association rate constant (k2) value for DS binding to 25% glycated HSA (k2 without FFA: 385 +/- 10 s-1, k2 with FFA > or = 385 +/- 10 s-1). In contrast, shorter chain fatty acids (hexanoic, octanoic, decanoic, lauric and myristic acids) showed marked inhibitory effects for 0.3 mmol/l FFA (k2 range: 233 +/- 32 to 69 +/- 5 s-1) and for 0.6 mmol/l FFA (k2 range: 125 +/- 3 to 20 +/- 4 s-1). The association rate constant (k2) as well as the affinity constant (KA) of DS were markedly affected by glycation: k2 was 686 +/- 61 s-1 for 7% glycated HSA, 385 +/- 10 s-1 for 25% glycated HSA and 209 +/- 12 s-1 for 50% glycated HSA. KA decreased from 6.1 +/- 2.9 x 10(5) M-1 for 7% glycated HSA, to 5.1 +/- 0.1 x 10(5) M-1 for 25% glycated HSA and to 1.3 +/- 0.6 x 10(5) M-1 for 50% glycated HSA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique, and pulse radiolysis

Flash photolysis of alkaline peroxynitrite solutions results in the formation of nitrogen monoxide and superoxide. From the rate of recombination it is concluded that the rate constant of the reaction of nitrogen monoxide with superoxide is (1.9 +/- 0.2) x 10(10) M-1 s-1. The pKa of hydrogen oxoperoxonitrate is dependent on the medium. With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations, while at 0.5 M phosphate the value is 7.5. The kinetics of decay do not follow first-order kinetics when the pH is larger than the pKa, combined with a total peroxynitrite and peroxynitrous acid concentration that exceeds 0.1 mM. An adduct between ONOO- and ONOOH is formed with a stability constant of (1.0 +/- 0.1) x 10(4) M. The kinetics of the decay of hydrogen oxoperoxonitrate are not very pressure-dependent: from stopped-flow experiments up to 152 MPa, an activation volume of 1.7 +/- 1.0 cm3 mol-1 was calculated. This small value is not compatible with homolysis of the O-O bond to yield free nitrogen dioxide and the hydroxyl radical. Pulse radiolysis of alkaline peroxynitrite solutions indicates that the hydroxyl radical reacts with ONOO- to form [(HO)ONOO].- with a rate constant of 5.8 x 10(9) M-1 s-1. This radical absorbs with a maximum at 420 nm (epsilon = 1.8 x 10(3) M-1 cm-1) and decays by second-order kinetics, k = 3.4 x 10(6) M-1 s-1. Improvements to the biomimetic synthesis of peroxynitrite with solid potassium superoxide and gaseous nitrogen monoxide result in higher peroxynitrite to nitrite yields than in most other syntheses.     

Thermodynamics of the interaction of the Escherichia coli regulatory protein TyrR with DNA studied by fluorescence spectroscopy

Fluorescence quenching was used to study the site-specific binding of the Escherichia coli regulatory protein TyrR to a fluoresceinated oligonucleotide (9F30A/30B) containing a TyrR binding site. The equilibrium constant for the interaction (KL) was measured as a function of temperature and salt concentration in the presence and absence of ATPgammaS, a specific ligand for TyrR. Fluorescence titrations yielded a KL value of 1.20 x 10(7) M-1 at 20 degrees C, which was independent of the acceptor (9F30A/30B) concentration in the range 5-500 nM, indicating that the system exhibits true equilibrium binding. Clarke and Glew analysis of the temperature dependence of binding revealed a linear dependence of R ln KL on temperature in the absence of ATPgammaS. The thermodynamic parameters obtained at 20 degrees C (theta) were = -35.73 kJ mol-1, = 57.41 kJ mol-1, and = 93.14 kJ mol-1. Saturating levels of ATPgammaS (200 microM) strengthened binding at all temperatures and resulted in a nonlinear dependence of Rln KL on temperature. The thermodynamic parameters characterizing binding under these conditions were = -39.32 kJ mol-1, = 37.16 kJ mol-1, = 76.40 kJ mol-1, and = -1.03 kJ mol-1 K-1. Several conclusions were drawn from these data. First, binding is entropically driven at 20 degrees C in both the presence and absence of ATPgammaS. This can partly be accounted for by counterions released from the DNA upon TyrR binding; in the absence of ATPgammaS and divalent cations, the TyrR-9F30A/30B interaction results in the release of two to three potassium ions. Second, the more favorable value, and hence tighter binding observed in the presence of ATPgammaS, is primarily due to a decrease in (-20.3 kJ mol-1), which overcomes an unfavorable decrease in (-16.7 kJ mol-1). Third, the negative value obtained in the presence of ATPgammaS indicates that the binding of ATPgammaS favors a conformational change in TyrR upon binding to 9F30A/30B, yielding a more stable complex.     

Kinetic mechanism of a partial folding reaction. 1. Properties Of the reaction and effects of denaturants

The bimolecular association rate constant (kon) and dissociation rate constant (koff) of the complex between fluorescein-labeled S-peptide analogues and folded S-protein are reported. This is the first kinetic study of a protein folding reaction in which most of the starting material is already folded and only a small part (one additional helix) becomes ordered; it provides a folding landscape with a small conformational entropy barrier, and one in which kinetic traps are unlikely. Refolding and unfolding are measured under identical strongly native conditions, and the reaction is found to be two-state at low reactant concentrations. The dissociation constant (Kd) of the complex and the properties of the transition state may be calculated from the rate constants without extrapolation. The folded complex is formed fast (kon = 1.8 x 10(7) M-1 s-1) and is very stable (Kd = 6 pM) at 10 degrees C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex by 1.4 kcal mol-1. The charge effect enters in the refolding reaction: increasing the salt concentration reduces kon dramatically and has little effect on koff. Urea and GdmCl destabilize the complex by decreasing kon and increasing koff. The slopes (m-values) of plots of ln Kd vs [cosolvent] are 0.75 +/- 0.04 and 2.8 +/- 0.3 kcal mol-1 M-1 for urea and GdmCl, respectively. The ratio mon/(mon + moff) is 0.54 +/- 0.04 for urea and 0.57 +/- 0.1 for GdmCl, where mon is the m-value for kon and moff is the m-value for koff, indicating that more than half of the sites for interaction with either cosolvent are buried in the ensemble of structures present at the transition state.     

Stability of recombinant yeast protoporphyrinogen oxidase: effects of diphenyl ether-type herbicides and diphenyleneiodonium

Protoporphyrinogen oxidase catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX and is the molecular target of diphenyl ether-type herbicides. Structural features of yeast protoporphyrinogen oxidase were assessed by circular dichroism studies on the enzyme purified from E. coli cells engineered to overproduce the protein. Coexpression of the bacterial gene ArgU that encodes tRNAAGA,AGG and a low induction temperature for protein synthesis were critical for producing protoporphyrinogen oxidase as a native, active, membrane-bound flavoprotein. The secondary structure of the protoporphyrinogen oxidase was 40.0 +/- 1. 5% alpha helix, 23.5 +/- 2.5% beta sheet, 18.0 +/- 2.0% beta turn, and 18.5 +/- 2.5% random-coil. Purified protoporphyrinogen oxidase appeared to be a monomeric protein that was relatively heat-labile (Tm of 44 +/- 0.5 degreesC). Acifluorfen, a potent inhibitor that competes with the tetrapyrrole substrate, and to a lower extent FAD, the cofactor of the enzyme, protected the protein from thermal denaturation, raising the Tm to 50.5 +/- 0.5 degreesC (acifluorfen) and 46.5 +/- 0.5 degreesC (FAD). However, diphenyleneiodonium, a slow tight-binding inhibitor that competes with dioxygen, did not protect the enzyme from heat denaturation. Acifluorfen binding to the protein increased the activation energy for the denaturation from 15 to 80 kJ.mol-1. The unfolding of the protein was a two-step process, with an initial fast reversible unfolding of the native protein followed by slow aggregation of the unfolded monomers. Functional analysis indicated that heat denaturation caused a loss of enzyme activity and of the specific binding of radiolabeled inhibitor. Both processes occurred in a biphasic manner, with a transition temperature of 45 degreesC.