FlgM proteins from different bacteria exhibit different structural characteristics

Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not require a stable structure for function. The importance of IDPs in many biological processes has been established but there remain unanswered questions about their evolution and conservation of their disordered state within a protein family. Our group has been studying the structural similarities among orthologous FlgM proteins, a model class of IDPs. We have previously shown that the FlgM protein from the thermophile Aquifex aeolicus has more structure at A. aeolicus' physiological temperature (85 °C) than is observed for the Salmonella typhimurium FlgM, suggesting that the disordered nature of FlgM varies among organisms and is not universally conserved. In this work, we extend these studies to the FlgM proteins from Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Bacillus subtilis. We demonstrate that the B. subtilis, E. coli, and S. typhimurium FlgMs exist in a premolten globule-like conformation, though the B. subtilis FlgM is in a more compacted conformation than the other two. The P. aeruginosa and P. mirabilis FlgM proteins exist in a currently unknown conformation that is not either coil-like or premolten globule-like. The P. aeruginosa FlgM appears to contain more weak intramolecular contacts given its more compacted state than the P. mirabilis FlgM. These results provide experimental evidence that members of the same protein family can exhibit different degrees of disorder, though understanding how different disordered states evolve in the same protein family will require more study.     

The Zinc Center Influences the Redox and Thermodynamic Properties of Escherichia coli Thioredoxin 2

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli cytoplasm contains two thioredoxins, Trx1 and Trx2. Trx2 is special because it is induced under oxidative stress conditions and it has an additional N-terminal zinc-binding domain. We have determined the redox potential of Trx2, the pK_a of the active site nucleophilic cysteine, as well as the stability of the oxidized and reduced form of the protein. Trx2 is more oxidizing than Trx1 (-221 mV versus -284 mV, respectively), which is in good agreement with the decreased value of the pK_a of the nucleophilic cysteine (5.1 versus 7.1, respectively). The difference in stability between the oxidized and reduced forms of an oxidoreductase is the driving force to reduce substrate proteins. This difference is smaller for Trx2 (ΔΔG°_H2O = 9 kJ/mol and ΔT_m = 7. 4 °C) than for Trx1 (ΔΔG°_H2O = 15 kJ/mol and ΔT_m = 13 °C). Altogether, our data indicate that Trx2 is a significantly less reducing enzyme than Trx1, which suggests that Trx2 has a distinctive function. We disrupted the zinc center by mutating the four Zn²⁺-binding cysteines to serine. This mutant has a more reducing redox potential (-254 mV) and the pK_a of its nucleophilic cysteine shifts from 5.1 to 7.1. The removal of Zn²⁺ also decreases the overall stability of the reduced and oxidized forms by 3.2 kJ/mol and 5.8 kJ/mol, respectively. In conclusion, our data show that the Zn²⁺-center of Trx2 fine-tunes the properties of this unique thioredoxin.     

Thermal dependence of cardiac SR Ca-ATPase from fish and mammals

The thermal sensitivity of metabolic performance in vertebrates requires a better understanding of the temperature sensitivity of cardiac function. The cardiac sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) is vital for excitation–contraction (E–C) coupling and intracellular Ca²⁺ homeostasis in heart cells. To better understand the thermal dependency of cardiac output in vertebrates, we present comparative analyses of the thermal kinetics properties of SERCA2 from ectothermic and endothermic vertebrates. We directly compare SR ventricular microsomal preparations using similar experimental conditions from sarcoplasmic reticulum isolated from cardiac tissues of mammals and fish. The experiments were designed to delineate the thermal sensitivity of SERCA2 and its role in thermal sensitivity Ca²⁺ uptake and E–C coupling. Ca²⁺ transport in the microsomal SR fractions from rabbit and bigeye tuna (Thunnus obesus) ventricles were temperature dependent. In contrast, ventricular SR preparations from coho salmon (Onchorhychus kisutch) were less temperature dependent and cold tolerant, displaying Ca²⁺ uptake as low as 5 °C. As a consequence, the Q₁₀ values in coho salmon were low over a range of different temperature intervals. Maximal Ca²⁺ transport activity for each species occurred in a different temperature range, indicating species-specific thermal preferences for SERCA2 activity. The mammalian enzyme displayed maximal Ca²⁺ uptake activity at 35 °C, whereas the fish (tuna and salmon) had maximal activity at 30 °C. At 35 °C, the rate of Ca²⁺ uptake catalyzed by the bigeye tuna SERCA2 decreased, but not the rate of ATP hydrolysis. In contrast, the salmon SERCA2 enzyme lost its activity at 35 °C, and ATP hydrolysis was also impaired. We hypothesize that SERCA2 catalysis is optimized for species-specific temperatures experienced in natural habitats and that cardiac aerobic scope is limited when excitation–contraction coupling is impaired at low or high temperatures due to loss of SERCA2 enzymatic function.     

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

Adduct formation between ternary Pt(II)-amino acid-aromatic diimine complexes and flavin mononucleotide and its effect on redox properties

Adduct formation of ternary Pt(II) complexes composed of an amino acid and an aromatic diimine, [Pt(A)(DA)] (A = glycinate (Gly), alaninate (Ala), valinate, or arginine (Arg); DA = 2,2′-bipyridine (bpy) or 1,10-phenanthroline (phen)), with flavin mononucleotide (FMN) and anthraquinone-2-sulfonate (AQS) were investigated by spectroscopic, X-ray diffraction, and electrochemical methods. The Pt(II) complexes formed 1:1 [Pt(A)(DA)]-FMN adducts by stacking with the aromatic moiety of FMN, and the stability constants, log K, for the systems with [Pt(A)(phen)] (A = Gly, Ala, and Arg) and [Pt(Arg)(bpy)] were determined to be 2.83(8)-3.42(6) from ¹H NMR spectra at 25 °C in D₂O (I = var.). The structure of the adduct [Pt(Ala)(phen)](AQS) (1) was determined by X-ray analysis to involve a π-π stacking interaction between coordinated phen and AQS with the distance of 3.400(7) Å and a hydrogen bond between the sulfonate moiety of AQS and the amino group of coordinated Ala. Cyclic voltammetry of the 1:1 [Pt(A)(DA)]-FMN systems in a phosphate buffer (pH 7.0) showed that the potentials, E_1/2, for the two-electron redox process of FMN shifted to higher values by 18-31 mV as compared with the value for free FMN.     

Simultaneous true, gated, and coupled electron-transfer reactions and energetics of protein rearrangement

Cytochromes c₆ and f react by three et mechanisms under similar conditions. We report temperature and viscosity effects on the protein docking and kinetics of ³Zncyt c₆ + cyt f(III) → Zncyt c₆⁺ + cyt f(II). At 0.5-40.0 °C, this reaction occurs within the persistent (associated) diprotein complex with the rate constant k^pr and within the transient (collision) complex with the rate constant k^tr. The viscosity independence of k^pr, the donor-acceptor coupling H_ab = (0.5 ± 0.1) cm⁻¹, and reorganizational energy λ = (2.14 ± 0.02) eV indicate true et within the persistent complex. The viscosity dependence of k^tr and a break at 30 °C in the Eyring plot for k^tr reveal mechanisms within the transient complex that are reversibly switched by temperature change. Kramers protein friction parameters differ much for the reactions below (σ = 0.3 ± 0.1, δ = 0.85 ± 0.07) and above (σ = 4.0 ± 0.9, δ = 0.40 ± 0.06) 30 °C. The transient complex(es) undergo(es) coupled et below ca. 30 °C and gated et above ca. 30 °C. Brownian dynamics simulations reveal two broad, dynamic ensembles of configurations “bridged” by few intermediate configurations through which the interconversion presumably occurs.     

Kinetics of the thermal inactivation and aggregate formation of rabbit muscle pyruvate kinase in the presence of trehalose

In a previous study we found that 30-40% dimethylsulfoxide induces the active conformation of rabbit muscle pyruvate kinase. Because dimethylsulfoxide is known to perturb structure and function of many proteins, we have explored the effect of trehalose on the kinetics of thermal inactivation and stability of pyruvate kinase; this is because trehalose, in contrast to dimethyl sulfoxide, is totally excluded from the hydration shell of proteins. The results show that 600 mM trehalose inhibits the activity of pyruvate kinase by about 20% at 25 °C, however, trehalose protects pyruvate kinase from thermal inactivation at 60 °C, increases the Tm_app of unfolding by 7.2 °C, induces a more compact state, and stabilizes its tetrameric structure. The inactivation process is irreversible due to the formation of protein aggregates. Trehalose diminishes the rate of formation of intermediates with propensity to aggregate, but does not affect the extent of aggregation. Remarkably, trehalose affects the aggregation process by inducing aggregates with amyloid-like characteristics.     

The Metalloregulatory Zinc Site in Streptococcus pneumoniae AdcR, a Zinc-activated MarR Family Repressor

Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain-heart infusion) under microaerobic conditions revealed upregulation of 13 genes, including adcR and adcCBA, encoding a high-affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and an approximately twofold increase in cell-associated Zn(II). Apo- and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K_DNA-Zn = 2.4 × 10⁸ M^- 1 (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with stepwise Zn(II) affinities K_Zn1 and K_Zn2 of ≥ 10⁹ M^- 1 at pH 6.0 and ≥ 10¹² M^- 1 at pH 8.0, and one to three lower affinity Zn(II) depending on the pH. X-ray absorption spectroscopy of the high-affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA-binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high-affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild type apo-AdcR, but fail to conformationally switch upon Zn(II) binding. These studies implicate His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.     

Inositol-1 (or 4)-monophosphatase from Glycine max embryo axes is a phosphatase with broad substrate specificity that includes phytate dephosphorylation

A phosphate-hydrolyzing activity from Glycine max embryo axes was purified by a series of chromatographic steps and electroelution from activity gels, and demonstrated to be an inositol-1 (or 4)-monophosphatase by partial internal amino acid sequence. This enzyme hydrolyzed ATP, sodium pyrophosphate (NaPPi), inositol hexakisphosphate, and inositol 1-monophosphate, but not p-nitrophenyl phosphate, ADP, AMP or glucose 6-P. Using NaPPi as substrate, the highly purified protein hydrolyzed up to 0.4 mmol phosphate min^− 1 mg^− 1 protein and had a Km_avg of 235 μM for NaPPi. Since NaPPi is relatively inexpensive and readily available, we used this as substrate for the subsequent characterization. We observed the following: (a) specific inhibition by Li and NaF but not by butanedione monoxime, or orthovanadate; (b) activation by Cu²⁺ and Mg²⁺; (c) optimum activity at pH 7.4; and (d) temperature stability after 1-h incubations at 37–80 °C, with maximum activity at 37 °C. The partially purified protein was detected by in-gel activity assays and the band was electroeluted to yield a highly purified protein. Analysis by SDS-PAGE and native IEF-PAGE yielded a single major polypeptide of 29 kDa and pI ∼ 5.9, respectively. In addition, in-gel activity from embryo axes and whole hypocotyls at early germination times revealed one high and one intermediate molecular weight isoform, but only the intermediate one corresponded to IMPase. Throughout the post-imbibition period, the activity of the high molecular weight isoform disappeared and IMPase increased, indicating an increasing expression of the enzyme as germination and growth proceeded. These data indicate that the inositol-1 (or 4)-monophosphatase present in the embryo axis of G. max has a wide phosphate substrate specificity, and may play an important role in phosphate metabolism during the germination process.     

Structural and magnetic characterization of a novel series of dinuclear Cu(II) complexes bridging by 2,5-pyrazine dicarboxylate

A new series of dinuclear 2,5-pyrazine dicarboxylato-bridged copper(II) complexes were synthesized and characterized by spectroscopic techniques. The complexes have the general structural formula [Cu₂(L)₂(μ-pyzdc)](ClO₄)₂·nH₂O where L = TPA, n = 2 (1); L = pmedien, n = 2 (2); L = aepn, n = 3 (3); L = dpt, n = 2 (4); L = Medpt, n = 0 (5); L = dien, n = 0 (6) and L = MeDPA, n = 2 (7) with TPA = tris(2-pyridylmethyl)amine, pmdien = N,N,N′,N′′,N′′-pentamethyldiethylenetriamine, aepn = N-(2-aminoethyl)-1,3-diaminopropane, dpt = dipropylene-triamine, Medpt = 3,3′-diamino-N-methyldipropylamine, dien = diethylenetriamine, MeDPA = N,N-di(2-pyridylmethyl)methylamine. In these complexes, the bridging nature of the 2,5-pyrazine dicarboxylato ligand (pyzdc) was confirmed by single-crystal X-ray crystallography. The structure of the TPA complex 1 consists of μ-pyzdc bridging two Cu(II) centers in a bis(monodentate) bonding fashion through a single oxygen atom supplied by each carboxylate group of the bridged pyzdc in a distorted trigonal bipyramidal geometry achieved by the four nitrogen atoms from the TPA ligand. In the complexes 2-5 derived from tridentate amines, the bridged pyzdc acts as a bis(bidentate) ligand in a distorted square pyramidal geometry achieved by one nitrogen and one carboxylate-oxygen of pyzdc, and by the three N-atoms of the amine coligands. The intradimer Cu?Cu distances in the complexes 2-5 are in the range 6.97-7.45 ? and in it is 10.96 ? in 1. The corresponding intermolecular distances are even shorter (5.34-7.99 ?). The susceptibility measurements at variable temperatures over the 5-300 K range reveal weak antiferromagnetic coupling with J values ranging from −0.61 to −4.78 cm⁻¹.     

Synthesis and characterization of quaternized β-chitin

Water-soluble 2′-O-hydroxypropyltrimethylammoniumchitin chloride (2′-O-HTACCt) was prepared directly from β-chitin and 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA) in basic medium. The effect of alkali concentration, reaction temperature, reaction time, and dosage of CTA on yield and degree of substitution (DS) of 2′-O-HTACCt were studied. These quaternized chitin derivatives were characterized by FTIR and ¹H NMR spectroscopy, conductometric titration, and elemental analysis methods. Research results indicate that β-chitin can react directly with CTA to produce a water-soluble 2′-O-HTACCt derivative with a high DS. The optimal preparation conditions were determined to be 35-40 wt % (aq NaOH), 40 °C (reaction temperature), 6 h (reaction time), and 4 (molar ratio of CTA to β-chitin unit).     

Probing excited states and activation energy for the integral membrane protein phospholamban by NMR CPMG relaxation dispersion experiments

Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca²⁺ ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca²⁺ATPase (SERCA). The PLN monomer is the functionally active form that exists in equilibrium between ordered (T state) and disordered (R state) states. While the T state has been fully characterized using a hybrid solution/solid-state NMR approach, the R state structure has not been fully portrayed. It has, however, been detected by both NMR and EPR experiments in detergent micelles and lipid bilayers. In this work, we quantitatively probed the μs to ms dynamics of the PLN excited states by observing the T state in DPC micelles using CPMG relaxation dispersion NMR spectroscopy under functional conditions for SERCA. The ¹⁵N backbone and ¹³C_δ1 Ile-methyl dispersion curves were fit using a two-state equilibrium model, and indicate that residues within domain Ia (residues 1-16), the loop (17-22), and domain Ib (23-30) of PLN undergo μs-ms dynamics (k_ex = 6100 ±800 s^- 1 at 17 °C). We measured k_ex at additional temperatures, which allowed for a calculation of activation energy equal to ∼ 5 kcal/mol. This energy barrier probably does not correspond to the detachment of the amphipathic domain Ia, but rather the energy needed to unwind domain Ib on the membrane surface, likely an important mechanism by which PLN converts between high and low affinity states for its binding partners.     

Engineering of a metagenome derived lipase toward thermal tolerance: effect of asparagine to lysine mutation on the protein surface

A highly thermostable mutant lipase was generated and characterized. Mutant enzyme demonstrated 144 fold enhanced thermostability over the wild type enzyme at 60 °C. Interestingly, the overall catalytic efficiency (k_cat/K_m) of mutant was also enhanced (~ 20 folds). Circular dichroism spectroscopy, studied as function of temperature, demonstrated that the mutant lipase retained its secondary structure up to 70-80 °C, whereas wild type protein structure was completely distorted above 35 °C. Additionally, the intrinsic tryptophan fluorescence (a probe for the tertiary structure) also displayed difference in the conformation of two enzymes during temperature dependent unfolding. Furthermore, mutation N355K resulted in extensive H-bonding (Lys355 HZ1OE2 Glu284) with a distance 2.44 Å. In contrast to this, Wt enzyme has not shown such H-bonding interaction.     

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 μM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 μM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 μM, respectively. The critical micellar concentration (CMC) of ODPC was 200 μM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T_c) of 41.5 °C and an enthalpy (?H) variation of 7.3 kcal mol⁻¹. The presence of 25 μM ODPC decreased T_c and ?H to 39.3 °C and 4.7 kcal mol⁻¹, respectively. ODPC at 250 μM destabilized the liposomes (36.3 °C, 0.46 kcal mol⁻¹). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.     

Electronic spectrum and magnetic properties of a dinuclear nickel(II) complex with two nickel(II) ions of C2-twisted octahedral geometry

A dinickel(II) complex [Ni₂(sym-hmp)₂](BPh₄)₂·3.5DMF·0.5(2-PrOH) (1) was synthesized with a dinucleating ligand, 2,6-bis[(2-hydroxyethyl)methylaminomethyl]-4-methyl-phenol [H(sym-hmp)]. The complex 1 (C₉₀H_118.50B₂N_7.50Ni₂O₁₀) crystallized in the triclinic space group with dimensions a = 14.7446(4) Å, b = 15.4244(4) Å, c = 18.7385(6) Å, α = 86.9495(9)°, β = 76.7263(10)°, γ = 86.5370(8)°, and V = 4136.8(2) Å³ and with Z = 2; this is isomorphous to a previous cobalt(II) complex [Co₂(sym-hmp)₂](BPh₄)₂. Single-crystal X-ray analysis revealed a bis(μ-phenoxo)dinickel(II) core structure containing two distorted octahedral nickel(II) ions of C₂ symmetry. The order of the coordination bond lengths is Ni-O(phenoxo) < Ni-O(hydroxy) < Ni-N. The electronic spectrum of 1 was typical for the octahedral nickel(II) complexes, but the axial elongation and the C₂-twist of the equatorial plane were found after a detailed analysis. The bond angles obtained by the electronic spectrum agreed with the crystallographically obtained bond angles within 2.3°. The order of the AOM parameters was e_{σ,O(phenoxo)} > e_{σ,O(hydroxy)} > e_σ,N, which was consistent with the order of the coordination bond lengths. Magnetic susceptibility data for 1 were fitted well with the parameters 2J = −69.7 cm⁻¹, D = 0.00 cm⁻¹, g = 2.17, and TIP = 265 × 10⁻⁶ cm³ mol⁻¹. The result indicates significant antiferromagnetic exchange interaction and negligible zero-field splitting, while the isostructural cobalt(II) complex showed an anisotropic behavior.     

Synthesis of new (σ-N,N′-diazadiene)(η-alkene)platinum(0) compounds

Novel, thermally stable, dark-red to orange Pt⁰(σ²-N,N′-diazadiene)(η²-alkene) compounds have been synthesized in good yields from Pt⁰(COD)₂ or Pt⁰(NBE)₃, by stepwise substitution of the respective dienes or alkenes by an electron-poor alkene (dimethyl fumarate, maleic anhydride or fumaronitrile), followed by the appropriate diazadiene ligand in dry diethyl ether at 20 °C (diazadiene=various N,N′-disubstitued-1,4-diaza-1,3-dienes). The complex Pt(DBA)₂ is less suited as a precursor for the synthesis of Pt⁰(σ²-N,N′-diazadiene)(η²-alkene) compounds. These zerovalent Pt(diazadiene)(η²-alkene) compounds constitute a useful category of starting materials for synthetic organoplatinum chemistry and catalysis.     

Effect of N-Guanyl Modifications on Early Steps in Catalysis of Polymerization by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W

Translesion DNA polymerases are more efficient at bypass of many DNA adducts than replicative polymerases. Previous work with the translesion polymerase Sulfolobus solfataricus Dpo4 showed a decrease in catalytic efficiency during bypass of bulky N²-alkyl guanine (G) adducts with N²-isobutylG showing the largest effect, decreasing ∼ 120-fold relative to unmodified deoxyguanosine (Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N²-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576). The effect of adduct size on individual catalytic steps has not been easy to decipher because of the difficulty of distinguishing early noncovalent steps from phosphodiester bond formation. We developed a mutant with a single Trp (T239W) to monitor fluorescence changes associated with a conformational change that occurs after binding a correct 2′-deoxyribonucleoside triphosphate (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) and, in the present work, utilized this approach to monitor insertion opposite N²-alkylG-modified oligonucleotides. We estimated maximal rates for the forward conformational step, which coupled with measured rates of product formation yielded rate constants for the conformational step (both directions) during insertion opposite several N²-alkylG adducts. With the smaller N²-alkylG adducts, the conformational rate constants were not changed dramatically (<  3-fold), indicating that the more sensitive steps are phosphodiester bond formation and partitioning into inactive complexes. With the larger adducts (≥  (2-naphthyl)methyl), the absence of fluorescence changes suggests impaired ability to undergo an appropriate conformational change, consistent with previous structural work.     

A cryoprotective and cold-adapted 1,3-β-endoglucanase from cherimoya (Annona cherimola) fruit

A 1,3-β-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO₂-treated cherimoya fruit (Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3-β-glucanase with a M_r of 22.07 kDa and a pI of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35 °C and it retained 40% maximum activity at 5 °C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50 °C. Kinetic studies revealed low k_cat (3.10 ± 0.04 s⁻¹) and K_m (0.32 ± 0.03 mg ml⁻¹) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k_cat at both 37 and 5 °C, and a low E_a (6.99 kJ mol⁻¹) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze-thaw inactivation of lactate dehydrogenase. Indeed, the PD₅₀ was 8.7 μg ml⁻¹ (394 nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO₂-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO₂.     

Selective binding of imatinib to the genetic variants of human α1-acid glycoprotein

Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia. Its strong plasma protein binding referred to α₁-acid glycoprotein (AGP) component was found to inhibit the pharmacological activity. AGP shows genetic polymorphism and the two main genetic variants have different drug binding properties. The binding characteristics of imatinib to AGP genetic variants and the possibility of its binding interactions were investigated by various methods. The results proved that binding of imatinib to the two main genetic variants is very different, the high affinity binding belongs dominantly to the F1-S variant. This interaction is accompanied with specific spectral changes (induced circular dichroism, UV change, intrinsic fluorescence quenching), suggesting that the bound ligand has chiral conformation that would largely overlap with other ligands inside the protein cavity. Binding parameters of K_a = 1.7(± 0.2) × 10⁶ M^− 1 and n = 0.94 could be determined for the binding on the F1-S variant at 37°. Imatinib binding on the A variant is weaker and less specific. The binding affinity of imatinib to human serum albumin (nK_a ≈ 3 × 10⁴ M^− 1) is low. Pharmacologically relevant binding interactions with other drugs can be expected on the F1-S variant of AGP.     

KatG from Salmonella Typhimurium is a peroxynitritase

Pathogenic bacteria elicit protective responses to oxidative and nitrosative stresses. Although such responses are generally distinct, it was recently reported in Mycobacterium tuberculosis that catalase-peroxidase (KatG), a classical defence against peroxides, also exhibits peroxynitritase activity. Here, the katG gene from Salmonella Typhimurium was cloned and protein purified and characterised. An increase in the rate of decomposition of peroxynitrite was observed for KatG from the enterobacterium with a second-order rate constant of 4.2 × 10⁴ M⁻¹ s⁻¹ at pH 7.4, 25 °C. This enzyme was able to reduce dihydrorhodamine oxidation by peroxynitrite to ∼83%. Given the peroxynitritase activity demonstrated here it is likely that KatG may play a wider role in the detoxification of oxidative stresses than previously thought.