The heterotrimer of the membrane-peripheral components of transhydrogenase and the alternating-site mechanism of proton translocation.

Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI(2)dIII(1) heterotrimer in solution, but we could find no evidence for the formation of a dI(2)dIII(2) tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotide-binding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI(2)dIII(1) heterotrimer were investigated. (a) The rate of release of NADP(+) from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI(2)dIII(1) complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.

Pressure cell assisted solution characterization of polysaccharides. 1. Guar gum.

To reduce time-dependent aggregation phenomena and achieve true "molecular" solution, the "pressure cell" solubilization method of Vorwerg and co-workers was applied to solutions of guar galactomannans (three samples of different molecular weights), using various heating, time, and pressure profiles. Physicochemical characterization of the guar samples before and after pressure cell treatment included measurements of intrinsic viscosity [eta] by capillary viscometry and M(w) and radius of gyration from size exclusion chromatography coupled to multiangle laser light scattering (SEC/MALLS). Heating the guar solutions (100-160 degrees C) without pressurization produced chain degradation with [eta] and M(w) values being reduced significantly, whereas this effect was reduced substantially for samples subject to initial pressurization ( approximately 5-10 bar). The constants in the Mark-Houwink-Sakurada equation, relating [eta] and M(w) were established and the characteristic ratio C(infinity) and chain persistence length L(p) were calculated using both the Burchard-Stockmayer-Fixman (BSF) method for flexible and semiflexible chains and the Hearst method more appropriate for stiffened chains. Definitive conclusions can now be drawn on the flexibility of the guar chain backbone, with L(p) approximately 4 nm from the BSF plot, in good agreement with previously published work using such geometric methods. This contrasts with the higher values obtained from extrapolation of data for polyelectrolytes with a similar backbone geometry, such as sodium carboxymethyl cellulose, to "infinite" ionic strength.

Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution.

BACKGROUND: The peroxiredoxins (Prxs) are an emerging family of multifunctional enzymes that exhibit peroxidase activity in vitro, and in vivo participate in a range of cellular processes known to be sensitive to reactive oxygen species. Thioredoxin peroxidase B (TPx-B), a 2-Cys type II Prx from erythrocytes, promotes potassium efflux and down-regulates apoptosis and the recruitment of monocytes by endothelial tissue. RESULTS: The crystal structure of human decameric TPx-B purified from erythrocytes has been determined to 1.7 [corrected)] A resolution. The structure is a toroid comprising five dimers linked end-on through predominantly hydrophobic interactions, and is proposed to represent an intermediate in the in vivo reaction cycle. In the crystal structure, Cys51, the site of peroxide reduction, is oxidised to cysteine sulphinic acid. The residue Cys172, lies approximately 10 A away from Cys51 [corrected]. CONCLUSIONS: The oxidation of Cys51 appears to have trapped the structure into a stable decamer, as confirmed by sedimentation analysis. A comparison with two previously reported dimeric Prx structures reveals that the catalytic cycle of 2-Cys Prx requires significant conformational changes that include the unwinding of the active-site helix and the movement of four loops. It is proposed that the stable decamer forms in vivo under conditions of oxidative stress. Similar decameric structures of TPx-B have been observed by electron microscopy, which show the protein associated with the erythrocyte membrane.

Conformational spectra--probing protein conformational changes.

Stafford [Biophys. J. 17 (1996) MP452] has shown that it is possible, using the analytical ultracentrifuge in sedimentation velocity mode, to calculate the molecular weights of proteins with a precision of approximately 5%, by fitting Gaussian distributions to g(s*) profiles so long as partial specific volume and the radial position of the meniscus are known. This makes possible the analysis of systems containing several components by the fitting of multiple distributions to the total g(s*) profile. We have found the Stafford relationship to hold for a range of protein solutes, particularly good agreement being found when the g(s*) profiles are computed from Schlieren (dc/dr vs. r) data using the Bridgman equation [J. Am. Chem. Soc. 64 (1942) 2349] . On this basis, we have developed a new approach to the analysis of systems where two or more distinguishable conformations of a single species are present, either in the same sample cell or in different cells in the same rotor. In the former case, this allows us to analyse a given solution of pure protein (i.e. monodisperse with respect to M) to reveal the presence in that solution of two or more conformers under identical solvent conditions. In the latter case, we can detect with high sensitivity any conformational change occurring in the transition from one set of solvent conditions to another. Alternatively, in this case, we can analyse slightly different proteins (e.g. deletion mutants) for conformational changes under identical solvent conditions. Examples of these procedures using well-defined protein systems are given.

Self-interaction of pneumolysin, the pore-forming protein toxin of Streptococcus pneumoniae.

The pathogenically important cholesterol-binding pore-forming bacterial "thiol-activated" toxins (TATs) are commonly believed to be monomeric in solution and to undergo a transition on membrane binding mediated by cholesterol to an oligomeric pore. We present evidence, gained through the application of a number of biochemical and biophysical techniques with associated modelling, that the TAT from Streptococcus pneumoniae, pneumolysin, is in fact able to self-associate in solution to form the same oligomeric structures. The weak interaction leading to solution oligomerization is manifested at low concentrations in a dimeric toxin form. The inhibition of toxin self-interaction by derivatization of the single cysteine residue in pneumolysin with the thiol-active agent dithio (bis)nitrobenzoic acid indicates that self-interaction is mediated by the fourth domain of the protein, which has a fold similar to other proteins known to self-associate. This interaction is thought to have implications for the understanding of mechanisms of pore formation and complement activation by pneumolysin.

Probing the stabilizing role of C-terminal residues in trimethylamine dehydrogenase.

In trimethylamine dehydrogenase, a homodimeric iron-sulfur flavoprotein, the C-terminal 17 residues of each subunit (residues 713-729) embrace residues on the other subunit. The role of this unusual mode of interaction at the subunit interface was probed by isolating three mutant forms of trimethylamine dehydrogenase in which the C-terminus of the enzyme was deleted by five residues [delta(725-729], 10 residues [delta(720-729)] and 17 residues [delta(713-729)]. The solution properties and conformational states of the three mutant enzymes were investigated using optical, fluorescence and circular dichroism spectroscopies, ANS binding and a novel and conformationally sensitive hydrodynamic method. The data reveal that sequential deletion of the C-terminus of trimethylamine dehydrogenase does not affect significantly dimer stability or the overall structural integrity of the enzyme. However, deletion of the C-terminus severely compromises, but does not abolish, the ability of the enzyme to become covalently coupled with the redox cofactor FMN in the active site, located over 20 A from the C-terminus. Hydrodynamic studies reveal minor conformational changes in the deletion mutants that lead to a more compact enzyme structure. These conformational changes are probably transmitted to the active site via altering the interaction of the C-terminus with the second helix in the beta/alpha barrel of trimethylamine dehydrogenase, leading to poor flavinylation during the folding of the enzyme and assembly with FMN.

A core-shell model of calcium phosphate nanoclusters stabilized by beta-casein phosphopeptides, derived from sedimentation equilibrium and small-angle X-ray and neutron-scattering measurements.

Calcium phosphate nanoclusters were prepared under standardised conditions using 10 mg ml(-1) of the 25-amino-acid N-terminal tryptic phosphopeptide of bovine beta-casein as a stabilising agent. The Mr determined by sedimentation equilibrium was 197,600+/-13,700 and the apparent radius of gyration determined by X-ray scattering was 2.80+/-0.05 nm. A small-angle neutron scattering contrast variation study in 1H2O/2H2O mixtures was performed and gave radii of gyration at the calculated match points for the calcium phosphate (88.2% 2H2O) and phosphopeptide (41.3% 2H2O) of 3.39+/-0.08 nm and 1.85+/-0.05 nm, respectively. Measurements at larger scattering wave vector showed a subsidiary maximum at about Q = 1.6 nm(-1). The results are consistent with a model of the nanoclusters comprising a spherical core of 355+/-20 CaHPO4 x 2 H2O units, density 2.31 g ml(-1) and radius 2.30+/-0.05 nm surrounded by 49+/-4 peptide chains with a partial specific volume of 0.7 cm3 g(-1), forming a tightly packed shell with an outer radius of 4.04+/-0.15 nm. This model suggests that the phosphopeptide is able to arrest the process of growth of the precipitating phase of calcium phosphate at its earliest stages. A similar role for whole casein could be vital to the normal functioning of the mammary gland during milk secretion.

Hydrodynamic characterization of chitosans varying in degree of acetylation.

The molecular weights and gross aqueous solution conformation of two chitosans of different degrees of acetylation, 'Sea Cure +210' (11% acetylated) and KN50 (58% acetylated) were characterized by viscometry, analytical ultracentrifugation and dynamic light scattering. The hydrodynamic parameters obtained were used to determine both the molecular weights and the gross solution conformation of the two chitosans. Using the Wales-Van Holde ratio of sedimentation coefficient concentration regression coefficient (ks) to intrinsic viscosity [eta], the Sea Cure +210 chitosan, which is much less acetylated than the KN50, is highly asymmetric in conformation. This, in conjunction with the charge on the molecule, would suggest a rod-like conformation in solution. The two largest KN50 chitosans have widely differing values for the Wales-Van Holde ratio, suggesting different solution conformation. However, when the series is examined as a whole using the Mark-Houwink-Kuhn-Sakurada relationships relating molecular weight to both intrinsic viscosity and translational diffusion coefficient, then a more spheroidal structure, approximating to a random coil, is predicted.

Minimum dose requirements of steroid-dependent asthmatic patients for aerosol beclomethasone and oral prednisone.

In 34 steroid-dependent asthma patients who improved markedly during 2 mo of treatment when progressively larger doses of beclomethasone aerosol were added to their oral prednisone regimen, we subsequently reduced both steroids to ascertain the minimum dose of each needed to prevent recurrence of significant asthmatic disability. After 80 wk of follow-up, 15 patients had successfully terminated oral prednisone; 19 were better controlled with a combination of aerosol plus oral steroid than with either drug alone; all patients previously unable to convert to alternate-day prednisone did so successfully during the combined therapy. The minimum effective maintenance dosage varied greatly among these patients-the median values being 2.5 mg prednisone and 1,200 microgram beclomethasone per day. The latter ranged from 200 to 1,8000 microgram. Only 4 patients were satisfactorily controlled without prednisone on 400 microgram beclomethasone per day or less. Seven needed extra intranasal beclomethasone to help control the nasal polyps which worsened after prednisone withdrawal. Suppression of plasma cortisol levels, apparently attributable to the beclomethasone, persisted in most patients, but on the average this was no worse than before commencing this treatment and valuable clinical improvement accrued. There were no other important complications of the regimen. In most of these patients with severe chronic asthma, optimum control of the disease required combined aerosol-oral therapy and maintenance doses of beclomethasone higher than those usually recommended. In some patients, effective control of chronic asthma by beclomethasone treatment may require acceptance of some persisting suppression of adrenal function as a considered risk.

A graded dose assessment of the efficacy of beclomethasone dipropionate aerosol for severe chronic asthma.

In a 26-wk double-blind controlled study of 34 patients whose asthma had been poorly controlled despite oral steroids, valuable clinical and pulmonary function improvement was derived by adding beclomethasone aerosol to the prednisone regimen. The amount of improvement correlated linearly with beclomethasone dosage over the range 200 to 1,600 microng/day. These patients required relatively high dosage. Success in achieving asymptomatic status was only 26% with the conventional 400 microng/day and 60% at 1,600 microng/day. Oropharyngeal candidiasis was also dose-related but did not prohibit the use of high-dosage beclomethasone. Respiratory infections, physical signs, blood glucose, and electrolytes were unaffected by the drug. A dose-related suppression of cortisol secretion was demonstrated, but about 1/4 of the group had normal plasma cortisol even at 1,600 microng/day plus the oral prednisone. An individualized risk-benefit assessment seems a better basis for choosing an optimal beclomethasone regimen for each patient than adherence to a conventionalized fixed dosage of 400 microng/day. This requires definition of: (1) a specific goal of treatment in the individual patient and the beclomethasone dosage required to achieve it; (2) the adrenocortical functional response of that particular patient to the desired dose of beclomethasone; and (3) the presence and degree of any dose-limiting constraints such as preexisting complications of steroid use.