Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

“Reduced minus oxidized” difference extinction coefficients Δ? in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1 ± 1.0 mM⁻¹ cm⁻¹ and 27.0 ± 1.0 mM⁻¹ cm⁻¹ were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E_m values at pH 6.5 of 244 ± 11 mV and −94 ± 21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q_B site and the microenvironment of the heme group of Cyt b559 are discussed.     

Thermodynamic and kinetic characterization of apoHmpH, a fast-folding bacterial globin

Despite the widespread presence of the globin fold in most living organisms, only eukaryotic globins have been employed as model proteins in folding/stability studies so far. This work introduces the first thermodynamic and kinetic characterization of a prokaryotic globin, that is, the apo form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli. This bacterial globin has a widely different sequence but nearly identical structure to its eukaryotic analogues. We show that apoHmpH is a well-folded monomeric protein with moderate stability at room temperature [apparent ΔG°_UN(w) = − 3.1 ± 0.3 kcal mol^− 1; m_UN = − 1.7 kcal mol^− 1 M^− 1] and predominant α-helical structure. Remarkably, apoHmpH is the fastest-folding globin known to date, as it refolds about 4- to 16-fold more rapidly than its eukaryotic analogues (e.g., sperm whale apomyoglobin and soybean apoleghemoglobin), populating a compact kinetic intermediate (β_I = 0.9 ± 0.2) with significant helical content. Additionally, the single Trp120 (located in the native H helix) becomes locked into a fully native-like environment within 6 ms, suggesting that this residue and its closest spatial neighbors complete their folding at ultrafast (submillisecond) speed. In summary, apoHmpH is a bacterial globin that shares the general folding scheme (i.e., a rapid burst phase followed by slower rate-determining phases) of its eukaryotic analogues but displays an overall faster folding and a kinetic intermediate with some fully native-like traits. This study supports the view that the general folding features of bacterial and eukaryotic globins are preserved through evolution while kinetic details differ.     

Growth and annual production of the brown alga Laminaria japonica (Phaeophyta, Laminariales) introduced into the Uwa Sea in southern Japan

The brown alga Laminaria japonica is distributed from southern Hokkaido to the northeastern Honshu in Japan. Recently, aquaculture of L. japonica has expanded to the southern coast of Japan and to China along the East China Sea. In order to elucidate the growth, biomass and productivity of L. japonica in a subtropical area, we cultivated and examined it in the Uwa Sea, in southwestern Japan over a period of 2 years. The seawater temperature ranged from 13.8 to 26.8 °C in 2001/2002 and from 13.1 to 27.2 °C in 2002/2003. In 2001/2002, the maximum density, maximum mean length and maximum mean wet wt. of L. japonica were 59.7 ± 28.0 ind. 50 cm^− 1 (mean ± S.D.), 187.5 ± 82.7 cm (360 cm in the largest individual) and 130.1 ± 94.6 g wet wt., respectively. In 2002/2003, these values were 94.7 ± 22.2 ind. 50 cm^− 1, 159.3 ± 74.4 cm (300 cm in the largest individual) and 95.2 ± 69.5 g wet wt., respectively. Thus, the length and weight increased when the density was low (2001/2002), and the length and weight decreased when the density was high (2002/2003). The maximum biomass was estimated to be 7200 ± 3400 g wet wt. 50 cm^− 1 in 2001/2002 and 7300 ± 2000 g wet wt. 50 cm^− 1 in 2002/2003. Annual production was estimated to be 33.3 kg wet wt. m^− 1 year^− 1 in 2001/2002 and 34.0 kg wet wt. m^− 1 year^− 1 in 2002/2003. The present study indicates that the annual production of L. japonica per rope of 1 m at Uwajima Bay, the Uwa Sea corresponded to 1.1-2.2 m² of that of Hokkaido in their native area. Thus, the present study indicates that L. japonica is highly adaptable because it is able to keep a high level of productivity when grown in water with a high temperature.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Downhill versus Barrier-Limited Folding of BBL 1: Energetic and Structural Perturbation Effects upon Protonation of a Histidine of Unusually Low pKa

A dispersion of melting temperatures at pH 5.3 for individual residues of the BBL protein domain has been adduced as evidence for barrier-free downhill folding. Other members of the peripheral subunit domain family fold cooperatively at pH 7. To search for possible causes of anomalies in BBL's denaturation behavior, we measured the pH titration of individual residues by heteronuclear NMR. At 298 K, the pK_a of His142 was close to that of free histidine at 6.47 ± 0.04, while that of the more buried His166 was highly perturbed at 5.39 ± 0.02. Protonation of His166 is thus energetically unfavorable and destabilizes the protein by ∼ 1.5 kcal/mol. Changes in C^α secondary shifts at pH 5.3 showed a decrease in helicity of the C-terminus of helix 2, where His166 is located, which was accompanied by a measured decrease of 1.1 ± 0.2 kcal/mol in stability from pH 7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only ∼ 1% of the structurally perturbed state will be present at the biologically relevant pH 7.6. Experiments at pH 5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pK_a induced by changes in temperature will have near-maximal effects on pH-dependent conformational equilibria and on propagation of experimental error. Accordingly, conventional barrier-limited folding predicts some dispersion of measured thermal unfolding curves of individual residues at pH 5.3.     

ESR study of sublevel structure of anionic bis(phthalocyaninato)gadolinium(III)

Sublevel structure of the ⁸S_7/2 electronic ground state of anionic bis(phthalocyaninato)gadolinium(III) has been determined by simulation analysis of an ESR spectrum in frozen solution. The simplex multidimensional minimization algorithm was employed to find the zero-field-splitting parameter set giving the minimum RMS error from the observed spectrum. The parameter set {B₂⁰,B₄⁰,B₆⁰} for the potential of D_4d symmetry has been determined to be ±{(1.54 ± 0.01)× 10⁻² cm⁻¹, (0.9 ± 0.1)× 10⁻⁴ cm⁻¹, (−0.6 ± 0.9)× 10⁻⁶ cm⁻¹}. The energy difference between the lowest and highest sublevels has been found to be about 0.5 cm⁻¹.     

Copper(II)-induced secondary structure changes and reduced folding stability of the prion protein

The cellular isoform of the prion protein PrP^C is a Cu²⁺-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrP^C conformation are intimately linked with disease pathogenesis, the effect of Cu²⁺ ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu²⁺ ions destabilise the native fold of PrP^C. The midpoint of the unfolding transition is reduced by 0.73 ± 0.07 M urea in the presence of 1 mol equiv of Cu²⁺. This equates to an appreciable difference in free energy of unfolding (2.02 ± 0.05 kJ mol^− 1 at the midpoint of unfolding). We relate Cu²⁺-induced changes in secondary structure for full-length PrP(23-231) to smaller Cu²⁺ binding fragments. In particular, Cu²⁺-induced structural changes can directly be attributed to Cu²⁺ binding to the octarepeat region of PrP^C. Furthermore, a β-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90-126) is due only to local Cu²⁺ coordination to the individual binding sites centred at His95 and His110. Cu²⁺ binding does not directly generate a β-sheet conformation within PrP^C; however, Cu²⁺ ions do destabilise the native fold of PrP^C and may make the transition to a misfolded state more favourable.     

DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA

Escherichia coli HU_αβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU_αβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 10⁶ M^− 1, and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na⁺. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 10⁵ M^− 1 for the 10 bp mode, 3.5 ± 1.4 × 10⁴ M^− 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.     

Effects on capacitance by overexpression of membrane proteins

Functional Channelrhodopsin-2 (ChR2) overexpression of about 10⁴ channels/μm² in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C_m). The C_m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C_m of 1.15 ± 0.08 μF/cm² in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79 ± 0.02 μF/cm²). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C_m (0.85 ± 0.07 μF/cm²) as compared to non-expressing control cells (0.70 ± 0.03 μF/cm²). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.     

Impaired cardiac mitochondrial function and contractile reserve following an acute exposure to environmental particulate matter

Background

It has been suggested that mitochondrial function plays a central role in cardiovascular diseases associated with particulate matter inhalation. The aim of this study was to evaluate this hypothesis, with focus on cardiac O₂ and energetic metabolism, and its impact over cardiac contractility.

Methods

Swiss mice were intranasally instilled with either residual oil fly ash (ROFA) (1.0 mg/kg body weight) or saline solution. After 1, 3 or 5 h of exposure, O₂ consumption was evaluated in heart tissue samples. Mitochondrial respiration, respiratory chain complexes activity, membrane potential and ATP content and production rate were assessed in isolated mitochondria. Cardiac contractile reserve was evaluated according to the Langendorff technique.

Results

Three hours after ROFA exposure, tissue O₂ consumption was significantly decreased by 35% (from 1180 ± 70 to 760 ± 60 ng-at O/min g tissue), as well as mitochondrial rest (state 4) and active (state 3) respiration, by 30 and 24%, respectively (control state 4: 88 ± 5 ng-at O/min mg protein; state 3: 240 ± 20 ng-at O/min mg protein). These findings were associated with decreased complex II activity, mitochondrial depolarization and deficient ATP production. Even though basal contractility was not modified (control: 75 ± 5 mm Hg), isolated perfused hearts failed to properly respond to isoproterenol in ROFA-exposed mice. Tissue O₂ consumption rates positively correlated with cardiac contractile state in controls (r² = 0.8271), but not in treated mice (r² = 0.1396).

General Significance

The present results show an impaired mitochondrial function associated with deficient cardiac contractility, which could represent an early cardiovascular alteration after the exposure to environmental particulate matter.     

Lactose Binding to Galectin-1 Modulates Structural Dynamics, Increases Conformational Entropy, and Occurs with Apparent Negative Cooperativity

Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K₁ = 21 ± 6 × 10³ M^− 1) than the second (K₂ = 4 ± 2 × 10³ M^− 1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K₁ = 20 ± 10 × 10³ M^− 1 and K₂ = 1.67 ± 0.07 × 10³ M^− 1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general.     

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D_phys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R_g) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R_g value extrapolated from kinetic R_g data to time zero, R_g,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R_g² was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R_g of the minor component of D_phys containing the non-native cis proline isomer (D_phys,cis) to be 25.7 ± 0.6 Å. Moreover, R_g of the major component of D_phys containing the native proline isomer (D_phys,tra) was estimated as 23.9 ± 0.2 Å based on R_g,0. Consequently, both components of the burst-phase intermediate of barnase (D_phys,tra and D_phys,cis) are still largely expanded. It was inferred that D_phys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.     

Characterization of Membrane Protein Interactions by Isothermal Titration Calorimetry

Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix–helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of − 4.61 ± 0.04 kcal/mol at bicelle conditions where the sampling of random helix–helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of > 1 kcal/mol was obtained from helix–helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500 ± 100 s^− 1 and 2.1 ± 0.1 s^− 1, respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.     

Mechanistic studies on ADAMTS13 catalysis

The zinc-protease a disintegrin-like and metalloprotease with thrombospondin type I repeats (ADAMTS13) cleaves the Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ peptide bond of von Willebrand factor (VWF), avoiding the accumulation of ultra large VWF multimers. Hydrolysis by ADAMTS13 of a VWF analog (Asp¹⁵⁹⁶-Arg¹⁶⁶⁸ peptide, fluorescence energy transfer substrate [FRETS]-VWF73) was investigated by a fluorescence quenching method (FRETS method) from 15°C to 45°C and pH values from 4.5 to 10.5. The catalysis was influenced by two ionizable groups, whose pK_a values were equal to 6.41 ± 0.08 (ionization enthalpy = 32.6 ± 1.7 kJ/mol) and 4 ± 0.1 (ionization enthalpy = 3.8 ± 0.4 kJ/mol), whereas these values were equal to 6 ± 0.1 and 4.1 ± 0.1, respectively, in Co²⁺-substituted ADAMTS13. The catalytic process of FRETS-VWF73 hydrolysis showed negative activation entropy (−144 kJ/mol), suggesting that the transition state becomes more ordered than the ground state of the reactants. The k_cat/K_m values were not linearly correlated with temperature, as expression of change of the kinetic “stickiness” of the substrate. The Met¹⁶⁰⁶-Arg¹⁶⁶⁸ peptide product acted as hyperbolic mixed-type inhibitor of FRETS-VWF73 hydrolysis. Asp¹⁶⁵³, Glu¹⁶⁵⁵, Glu¹⁶⁶⁰, Asp¹⁶⁶³, together with the hydrophilic side chain of Thr¹⁶⁵⁶ were shown to form a “hot spot” in the VWF A2 sequence, which drives the molecular recognition and allosteric regulation of binding to ADAMTS13. The interaction of the Met¹⁶⁰⁶-Arg¹⁶⁶⁸ region of VWF with ADAMTS13 involves basic residues of the protease and is thus progressively inhibited at pH values >8.50. A molecular model of the FRETS-VWF73 showed that the substrate can fit into the active site only if ADAMTS13 assumes a C-like shape and, interacting with the acidic 1653-1668 region of VWF, properly orients the Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ peptide bond for the cleavage by the zinc-aquo complex in the active site.     

N-acylhydrazone improves exercise intolerance in rats submitted to myocardial infarction by the recovery of calcium homeostasis in skeletal muscle

Aims

This work investigated the effects of 3,4-methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294) treatment on the contractile response of soleus (SOL) muscle from rats submitted to myocardial infarction (MI).

Main methods

Following coronary artery ligation, LASSBio-294 (2 mg/kg, i.p.) or vehicle was administrated once daily for 4 weeks.

Key findings

The run time to fatigue for sham rats was 17.9 ± 2.6 min, and it was reduced to 3.3 ± 0.8 min (P < 0.05) in MI rats. In MI rats treated with LASSBio-294, the time to fatigue was 15.1 ± 3.6 min. During the contractile test, SOL muscles from sham rats showed a response of 7.12 ± 0.54 N/cm² at 60 Hz, which was decreased to 5.45 ± 0.49 N/cm² (P < 0.05) in MI rats. The contractility of SOL muscles from the MI-LASSBio-294 group was increased to 9.01 ± 0.65 N/cm². At 16 mM caffeine, the contractility was reduced from 2.31 ± 0.33 to 1.60 ± 0.21 N/cm² (P < 0.05) in the MI group. In SOL muscles from MI-LASSBio-294 rats, the caffeine response was increased to 2.62 ± 0.33 N/cm². Moreover, SERCA2a expression in SOL muscles was decreased by 0.31-fold (31%) in the MI group compared to the Sham group (P < 0.05). In the MI-LASSBio-294 group, it was increased by 1.53-fold (153%) compared to the MI group (P < 0.05). Meanwhile, the nuclear density in SOL muscles was increased in the MI group compared to the Sham group. Treatment with LASSBio-294 prevented this enhancement of cellular infiltrate.

Significance

LASSBio-294 treatment prevented the development of muscular fatigue and improved exercise intolerance in rats submitted to MI.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

The Contribution of a Covalently Bound Cofactor to the Folding and Thermodynamic Stability of an Integral Membrane Protein

The factors controlling the stability, folding, and dynamics of integral membrane proteins are not fully understood. The high stability of the membrane protein bacteriorhodopsin (bR), an archetypal member of the rhodopsin photoreceptor family, has been ascribed to its covalently bound retinal cofactor. We investigate here the role of this cofactor in the thermodynamic stability and folding kinetics of bR. Multiple spectroscopic probes were used to determine the kinetics and energetics of protein folding in mixed lipid/detergent micelles in the presence and absence of retinal. The presence of retinal increases extrapolated values for the overall unfolding free energy from 6.3 ± 0.4 kcal mol^− 1 to 23.4 ± 1.5 kcal mol^− 1 at zero denaturant, suggesting that the cofactor contributes 17.1 kcal mol^− 1 towards the overall stability of bR. In addition, the cooperativity of equilibrium unfolding curves is markedly reduced in the absence of retinal with overall m-values decreasing from 31.0 ± 2.0 kcal mol^− 1 to 10.9 ± 1.0 kcal mol^− 1, indicating that the folded state of the apoprotein is less compact than the equivalent for the holoprotein. This change in the denaturant response means that the difference in the unfolding free energy at a denaturant concentration midway between the two unfolding curves is only ca 3-6 kcal mol^− 1. Kinetic data show that the decrease in stability upon removal of retinal is associated with an increase in the apparent intrinsic rate constant of unfolding, k_u^H2O, from ~1 × 10^− 16 s^− 1 to ~1 × 10^− 4 s^− 1 at 25 °C. This correlates with a decrease in the unfolding activation energy by 16.3 kcal mol^− 1 in the apoprotein, extrapolated to zero SDS. These results suggest that changes in bR stability induced by retinal binding are mediated solely by changes in the activation barrier for unfolding. The results are consistent with a model in which bR is kinetically stabilized via a very slow rate of unfolding arising from protein-retinal interactions that increase the rigidity and compactness of the polypeptide chain.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

Germination characteristics and sporeling success of Azolla filiculoides Lamarck, an aquatic invasive fern, in a Mediterranean temporary wetland

Spore germination characteristics of Azolla filiculoides Lamarck, a recent invader of the Doñana Mediterranean wetlands, have been quantified experimentally. Batches of spores were subjected to different storage conditions, salinity concentrations, phosphorus availability and drought effects. Cold pre-treatment did not stimulate spore germination, whereas drought, a characteristic different from Azolla's native habitat, reduced it (40 ± 7 vs 13 ± 5). Germination declined with the increase of salinity (r = −0.50, notably above 5000 μS cm⁻¹), whereas phosphorus availability had no significant effect.