Increased folding stability of TEM-1 beta-lactamase by in vitro selection

In vitro selections of stabilized proteins lead to more robust enzymes and, at the same time, yield novel insights into the principles of protein stability. We employed Proside, a method of in vitro selection, to find stabilized variants of TEM-1 β-lactamase from Escherichia coli. Proside links the increased protease resistance of stabilized proteins to the infectivity of a filamentous phage. Several libraries of TEM-1 β-lactamase variants were generated by error-prone PCR, and variants with increased protease resistance were obtained by raising temperature or guanidinium chloride concentration during proteolytic selections. Despite the small size of phage libraries, several strongly stabilizing mutations could be obtained, and a manual combination of the best shifted the profiles for thermal unfolding and temperature-dependent inactivation of β-lactamase by almost 20 °C to a higher temperature. The wild-type protein unfolds in two stages: from the native state via an intermediate of the molten-globule type to the unfolded form. In the course of the selections, the native protein was stabilized by 27 kJ mol^− 1 relative to the intermediate and the cooperativity of unfolding was strongly increased. Three of our stabilizing replacements (M182T, A224V, and R275L) had been identified independently in naturally occurring β-lactamase variants with extended substrate spectrum. In these variants, they acted as global suppressors of destabilizations caused by the mutations in the active site. The comparison between the crystal structure of our best variant and the crystal structure of the wild-type protein indicates that most of the selected mutations optimize helices and their packing. The stabilization by the E147G substitution is remarkable. It removes steric strain that originates from an overly tight packing of two helices in the wild-type protein. Such unfavorable van der Waals repulsions are not easily identified in crystal structures or by computational approaches, but they strongly reduce the conformational stability of a protein.     

Complete Genomic Structure of the Cultivated Rice Endophyte Azospirillum sp. B510

We determined the nucleotide sequence of the entire genome of a diazotrophic endophyte, Azospirillum sp. B510. Strain B510 is an endophytic bacterium isolated from stems of rice plants (Oryza sativa cv. Nipponbare). The genome of B510 consisted of a single chromosome (3 311 395 bp) and six plasmids, designated as pAB510a (1 455 109 bp), pAB510b (723 779 bp), pAB510c (681 723 bp), pAB510d (628 837 bp), pAB510e (537 299 bp), and pAB510f (261 596 bp). The chromosome bears 2893 potential protein-encoding genes, two sets of rRNA gene clusters (rrns), and 45 tRNA genes representing 37 tRNA species. The genomes of the six plasmids contained a total of 3416 protein-encoding genes, seven sets of rrns, and 34 tRNAs representing 19 tRNA species. Eight genes for plasmid-specific tRNA species are located on either pAB510a or pAB510d. Two out of eight genomic islands are inserted in the plasmids, pAB510b and pAB510e, and one of the islands is inserted into trnfM-CAU in the rrn located on pAB510e. Genes other than the nif gene cluster that are involved in N₂ fixation and are homologues of Bradyrhizobium japonicum USDA110 include fixABCX, fixNOQP, fixHIS, fixG, and fixLJK. Three putative plant hormone-related genes encoding tryptophan 2-monooxytenase (iaaM) and indole-3-acetaldehyde hydrolase (iaaH), which are involved in IAA biosynthesis, and ACC deaminase (acdS), which reduces ethylene levels, were identified. Multiple gene-clusters for tripartite ATP-independent periplasmic-transport systems and a diverse set of malic enzymes were identified, suggesting that B510 utilizes C₄-dicarboxylate during its symbiotic relationship with the host plant.     

Phylogenetic reconstruction at the species and intraspecies levels in the genus Pisum (L.) (peas) using a histone H1 gene

A phylogenetic analysis of the genus Pisum (peas), embracing diverse wild and cultivated forms, which evoke problems with species delimitation, was carried out based on a gene coding for histone H1, a protein that has a long and variable functional C-terminal domain. Phylogenetic trees were reconstructed on the basis of the coding sequence of the gene His5 of H1 subtype 5 in 65 pea accessions. Early separation of a clear-cut wild species Pisum fulvum is well supported, while cultivated species Pisum abyssinicum appears as a small branch within Pisum sativum. Another robust branch within P. sativum includes some wild and almost all cultivated representatives of P. sativum. Other wild representatives form diverse but rather subtle branches. In a subset of accessions, PsbA-trnH chloroplast intergenic spacer was also analysed and found less informative than His5. A number of accessions of cultivated peas from remote regions have a His5 allele of identical sequence, encoding an electrophoretically slow protein product, which earlier attracted attention as likely positively selected in harsh climate conditions. In PsbA-trnH, a 8bp deletion was found, which marks cultivated representatives of P. sativum.     

Stabilizing roles of residual structure in the empty heme binding pockets and unfolded states of microsomal and mitochondrial apocytochrome b5

The microsomal (Mc) and mitochondrial (OM) isoforms of mammalian cytochrome b5 are the products of different genes, which likely arose via duplication of a primordial gene and subsequent functional divergence. Despite sharing essentially identical folds, heme-polypeptide interactions are stronger in OM b5s than in Mc b5s due to the presence of two conserved patches of hydrophobic amino acid side chains in the OM heme binding pockets. This is of fundamental interest in terms of understanding heme protein structure-function relationships, because stronger heme-polypeptide interactions in OM b5s in comparison to Mc b5s may represent a key source of their more negative reduction potentials. Herein we provide evidence that interactions amongst the amino acid side chains contributing to the hydrophobic patches in rat OM (rOM) b5 persist when heme is removed, rendering the empty heme binding pocket of rOM apo-b5 more compact and less conformationally dynamic than that in bovine Mc (bMc) apo-b5. This may contribute to the stronger heme binding by OM apo-b5 by reducing the entropic penalty associated with polypeptide folding. We also show that when bMc apo-b5 unfolds it adopts a structure that is more compact and contains greater nonrandom secondary structure content than unfolded rOM apo-b5. We propose that a more robust beta-sheet in Mc apo-b5s compensates for the absence of the hydrophobic packing interactions that stabilize the heme binding pocket in OM apo-b5s.     

Effects of N-acylethanolamines on the respiratory chain and production of reactive oxygen species in heart mitochondria

Long-chain N-acylethanolamines (NAEs) have been found to uncouple oxidative phosphorylation and to inhibit uncoupled respiration of rat heart mitochondria [Wasilewski, M., Wieckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Biochim. Biophys. Acta 1657, 151-163]. The aim of the present work was to investigate in more detail the mechanism of the inhibitory effects of NAEs on the respiratory chain. In connection with this, we also investigated a possible action of NAEs on the generation of reactive oxygen species (ROS) by respiring rat heart mitochondria. It was found that unsaturated NAEs, N-oleoylethanolamine (N-Ole) and, to a greater extent, N-arachidonoylethanolamine (N-Ara), inhibited predominantly complex I of the respiratory chain, with a much weaker effect on complexes II and III, and no effect on complex IV. Saturated N-palmitoylethanolamine had a much smaller effect compared to unsaturated NAEs. N-Ara and N-Ole were found to decrease ROS formation, apparently due to their uncoupling action. However, under specific conditions, N-Ara slightly but significantly stimulated ROS generation in uncoupled conditions, probably due to its inhibitory effect on complex I. These results may contribute to our better understanding of physiological roles of NAEs in protection against ischemia and in induction of programmed cell death.     

Structure and phase behavior of O-stearoylethanolamine: A combined calorimetric, spectroscopic and X-ray diffraction study

Recent studies show that O-acylethanolamines (OAEs), structural isomers of the putative stress-fighting lipids, namely N-acylethanolamines (NAEs), can be derived from NAEs and are present in biological membranes under physiological conditions. In view of this, we have synthesized O-stearoylethanolamine (OSEA) as a representative OAE and investigated its phase behavior and crystal structure. The thermotropic phase transitions of OSEA dispersed in water and in 150 mM NaCl were characterized using calorimetric, spectroscopic, turbidimetric and X-ray diffraction studies. These studies have revealed that when dispersed in water OSEA undergoes a cooperative phase transition centered at 53.8 °C from an ordered gel phase to a micellar structure whereas in presence of 150 mM NaCl the transition temperature increases to 55.8 °C and most likely the bilayer structure is retained above the phase transition. O-Stearoylethanolamine crystallized in the orthorhombic space group P2₁2₁2₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that OSEA molecules adopt a linear structure with all-trans conformation in the acyl chain region. The molecules are organized in a tail-to-tail fashion, similar to the arrangement in a bilayer membrane. These studies are relevant to understanding the role of salt on the phase properties of this new class of lipids.     

Molecular Determinants of a Native-State Prolyl Isomerization

Prolyl cis/trans isomerizations determine the rates of many protein-folding reactions, and they can serve as molecular switches and timers. The energy required to shift the prolyl cis/trans equilibrium during these processes originates from conformational reactions that are linked structurally and energetically with prolyl isomerization. We used the N2 domain of the gene-3-protein of phage fd to elucidate how such an energetic linkage develops in the course of folding. The Asp160-Pro161 bond at the tip of a β hairpin of N2 is cis in the crystal structure, but in fact, it exists as a mixture of conformers in folded N2. During refolding, about 10 kJ mol^− 1 of conformational energy becomes available for a 75-fold shift of the cis/trans equilibrium constant at Pro161, from 7/93 in the unfolded to 90/10 in the folded form. We combined single- and double-mixing kinetic experiments with a mutational analysis to identify the structural origin of this proline shift energy and to elucidate the molecular path for the transfer of this energy to Pro161. It originates largely, if not entirely, from the two-stranded β sheet at the base of the Pro161 hairpin. The two strands improve their stabilizing interactions when Pro161 is cis, and this stabilization is propagated to Pro161, because the connector peptides between the β strands and Pro161 are native-like folded when Pro161 is cis. In the presence of a trans-Pro161, the connector peptides are locally unfolded, and thus, Pro161 is structurally and energetically uncoupled from the β sheet. Such interrelations between local folding and prolyl isomerization and the potential modulation by prolyl isomerases might also be used to break and reestablish slow communication pathways in proteins.     

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Dihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (–CO–NH–) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl-β-amino acids. This enzyme is an attractive candidate for commercial production of D-amino acids, which are used in the production of semi-synthetic β-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH ∼ 6.0, which is stable up to pH ∼ 9.0, as concluded from fluorescence and CD. Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure.     

Thermodynamic properties of BPTI variants with highly simplified amino acid sequences

We report the first detailed thermodynamic analysis of simplified proteins by differential scanning calorimetry (DSC). The experiments were carried out with five simplified BPTI variants, whose structures and activities have been reported, in which several residues not essential for specifying the tertiary structure were replaced by alanine. In most aspects, the thermodynamics of simplified proteins were very similar to, if not essentially identical with, those of natural proteins. In particular, they undergo a highly cooperative two-state thermal unfolding process with a large enthalpy change, which is a thermodynamic hallmark of the native state of natural globular proteins. Furthermore, the specific enthalpy and entropy changes upon unfolding at 110 degrees C were close to values invariably observed for small natural globular proteins (55 J g(-1) and ~16 J K(-1) g(-1), respectively). On the other hand, two simplified BPTI variants, BPTI-21 and BPTI-22 (containing 21 and 22 alanine residues), were enthalpically stabilized while entropically destabilized with respect to the reference BPTI-[5,55] molecule. This peculiar type of entropy-enthalpy compensation is in sharp contrast to the usual enthalpy destabilization/entropy stabilization observed in mutational studies of natural proteins. Overall, we conclude that a thermodynamic native state can be achieved by proteins encoded with extensively simplified sequences.     

Explaining the geographical origins of seasonal influenza A (H3N2)

Most antigenically novel and evolutionarily successful strains of seasonal influenza A (H3N2) originate in East, South and Southeast Asia. To understand this pattern, we simulated the ecological and evolutionary dynamics of influenza in a host metapopulation representing the temperate north, tropics and temperate south. Although seasonality and air traffic are frequently used to explain global migratory patterns of influenza, we find that other factors may have a comparable or greater impact. Notably, a region''s basic reproductive number (R₀) strongly affects the antigenic evolution of its viral population and the probability that its strains will spread and fix globally: a 17–28% higher R₀ in one region can explain the observed patterns. Seasonality, in contrast, increases the probability that a tropical (less seasonal) population will export evolutionarily successful strains but alone does not predict that these strains will be antigenically advanced. The relative sizes of different host populations, their birth and death rates, and the region in which H3N2 first appears affect influenza''s phylogeography in different but relatively minor ways. These results suggest general principles that dictate the spatial dynamics of antigenically evolving pathogens and offer predictions for how changes in human ecology might affect influenza evolution.     

The structure of human apolipoprotein E2, E3 and E4 in solution. 2. Multidomain organization correlates with the stability of apoE structure

The stabilities toward thermal and chemical denaturation of three recombinant isoforms of human apolipoprotein E (r-apoE2, r-apoE3 and r-apoE4), human plasma apoE3, the recombinant amino-terminal (NT) and the carboxyl-terminal (CT) domains of plasma apoE3 at pH 7 were studied using near and far ultraviolet circular dichroism (UV CD), fluorescence and size-exclusion chromatography. By far UV CD, thermal unfolding was irreversible for the intact apoE isoforms and consisted of a single transition. The r-apoE3 was found to be less stable as compared to the plasma protein and the stability of recombinant isoforms was r-apoE4相似文献   

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.     

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

Isotopic signatures of N2O produced by ammonia-oxidizing archaea from soils

N₂O gas is involved in global warming and ozone depletion. The major sources of N₂O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N₂O produced by soil AOA and associated N₂O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N₂O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), δ¹⁵N^bulk and δ¹⁸O -N₂O of soil AOA strains were 13–30%, −13 to −35% and 22–36%, respectively, and strains MY1–3 and other soil AOA strains had distinct isotopic signatures. A ¹⁵N-NH₄⁺-labeling experiment indicated that N₂O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N₂O from AOA may be attributable to the relative contributions of these two processes. The highest N₂O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N₂O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N₂O emissions from soil nitrification may be attributable to AOA.     

Resveratrol induces cellular senescence with attenuated mono-ubiquitination of histone H2B in glioma cells

Resveratrol (3,4′,5-trihydroxy-trans-stilbene), a polyphenol naturally occurring in grapes and other plants, has cancer chemo-preventive effects and therapeutic potential. Although resveratrol modulates multiple pathways in tumor cells, how resveratrol or its affected pathways converge on chromatin to mediate its effects is not known. Using glioma cells as a model, we showed here that resveratrol inhibited cell proliferation and induced cellular hypertrophy by transforming spindle-shaped cells to enlarged, irregular and flatten-shaped ones. We further showed that resveratrol-induced hypertrophic cells expressed senescence-associated-β-galactosidase, suggesting that resveratrol-induced cellular senescence in glioma cells. Consistent with these observations, we demonstrated that resveratrol inhibited clonogenic efficiencies in vitro and tumor growth in a xenograft model. Furthermore, we found that acute treatment of resveratrol inhibited mono-ubiquitination of histone H2B at K120 (uH2B) in breast, prostate, pancreatic, lung, brain tumor cells as well as primary human cells. Chronic treatment with low doses of resveratrol also inhibited uH2B in the resveratrol-induced senescent glioma cells. Moreover, we showed that depletion of RNF20, a ubiquitin ligase of histone H2B, inhibited uH2B and induced cellular senescence in glioma cells in vitro, thereby recapitulated the effects of resveratrol. Taken together, our results suggest that uH2B is a novel direct or indirect chromatin target of resveratrol and RNF20 plays an important role in inhibiting cellular senescence programs that are intact in glioma cells.     

Molecular origin of the pH dependence of tyrosine D oxidation kinetics and radical stability in photosystem II

A role for redox-active tyrosines has been demonstrated in many important biological processes, including water oxidation carried out by photosystem II (PSII) of oxygenic photosynthesis. The rates of tyrosine oxidation and reduction and the Tyr/Tyr reduction potential are undoubtedly controlled by the immediate environment of the tyrosine, with the coupling of electron and proton transfer, a critical component of the kinetic and redox behavior. It has been demonstrated by Faller et al. that the rate of oxidation of tyrosine D (Tyr_D) at room temperature and the extent of Tyr_D oxidation at cryogenic temperatures, following flash excitation, dramatically increase as a function of pH with a pK_a of ≈ 7.6 [Faller et al. 2001 Proc. Natl. Acad. Sci. USA 98, 14368-14373; Faller et al. 2001 Biochemistry 41, 12914-12920]. In this work, we investigated, using FTIR difference spectroscopy, the mechanistic reasons behind this large pH dependence. These studies were carried out on Mn-depleted PSII core complexes isolated from Synechocystis sp. PCC 6803, WT unlabeled and labeled with ¹³C₆-, or ¹³C₁(4)-labeled tyrosine, as well as on the D2-Gln164Glu mutant. The main conclusions of this work are that the pH-induced changes involve the reduced Tyr_D state and not the oxidized Tyr_D state and that Tyr_D does not exist in the tyrosinate form between pH 6 and 10. We can also exclude a change in the protonation state of D2-His189 as being responsible for the large pH dependence of Tyr_D oxidation. Indeed, our data are consistent with D2-His189 being neutral both in the Tyr_D and Tyr_D states in the whole pH6-10 range. We show that the interactions between reduced Tyr_D and D2-His189 are modulated by the pH. At pH greater than 7.5, the ν(CO) mode frequency of Tyr_D indicates that Tyr_D is involved in a strong hydrogen bond, as a hydrogen bond donor only, in a fraction of the PSII centers. At pH below 7.5, the hydrogen-bonding interaction formed by Tyr_D is weaker and Tyr_D could be also involved as a hydrogen bond acceptor, according to calculations performed by Takahashi and Noguchi [J. Phys. Chem. B 2007 111, 13833-13844]. The involvement of Tyr_D in this strong hydrogen-bonding interaction correlates with the ability to oxidize Tyr_D at cryogenic temperatures and rapidly at room temperature. A strong hydrogen-bonding interaction is also observed at pH 6 in the D2-Gln164Glu mutant, showing that the residue at position D2-164 regulates the properties of Tyr_D. The IR data point to the role of a protonatable group(s) (with a pK_a of ≈ 7) other than D2-His189 and Tyr_D, in modifying the characteristics of the Tyr_D hydrogen-bonding interactions, and hence its oxidation properties. It remains to be determined whether the strong hydrogen-bonding interaction involves D2-His189 and if Tyr_D oxidation involves the same proton transfer route at low and at high pH.     

The cap binding complex influences H2B ubiquitination by facilitating splicing of the SUS1 pre-mRNA

Pre-messenger RNA splicing is carried out by a large ribonucleoprotein complex called the spliceosome. Despite the striking evolutionary conservation of the spliceosomal components and their functions, controversy persists about the relative importance of splicing in Saccharomyces cerevisiae—particularly given the paucity of intron-containing genes in yeast. Here we show that splicing of one pre-messenger RNA, SUS1, a component of the histone H2B ubiquitin protease machinery, is essential for establishing the proper modification state of chromatin. One protein complex that is intimately involved in pre-mRNA splicing, the yeast cap-binding complex, appears to be particularly important, as evidenced by its extensive and unique genetic interactions with enzymes that catalyze histone H2B ubiquitination. Microarray studies show that cap binding complex (CBC) deletion has a global effect on gene expression, and for ∼20% of these genes, this effect is suppressed when ubiquitination of histone H2B is eliminated. Consistent with this finding of histone H2B dependent effects on gene expression, deletion of the yeast cap binding complex leads to overubiquitination of histone H2B. A key component of the ubiquitin-protease module of the SAGA complex, Sus1, is encoded by a gene that contains two introns and is misspliced when the CBC is deleted, leading to destabilization of the ubiquitin protease complex and defective modulation of cellular H2B levels. These data demonstrate that pre-mRNA splicing plays a critical role in histone H2B ubiquitination and that the CBC in particular helps to establish the proper state of chromatin and proper expression of genes that are regulated at the level of histone H2B ubiquitination.