Amino acid substitutions in the ε-subunit of the F1F0-ATPase of Escherichia coli

A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature ε-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp. This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton translocation and lowered oxidative phosphorylation, but did not affect proton translocation through the F₀. Purified F₁-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal F₁-ATPase. Partial revertant strains were isolated in which Pro-47 of the ε-subunit was replaced by Ser or Thr. Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II β-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III β-turn. Space-filling models of the β-turn (residues 46–49) in the normal, mutant and partial revertant ε-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position. It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the ε-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the F₁-ATPase.     

Development of polarity in the ovules of the F2-progeny of theOenothera hybridalbicans · hhookeri

In the F₂-progeny of hybrids from crosses betweenOenothera biennis orsuaveolens andOe. hookeri with theRenner-complexesalbicans and^hhookeri, the development of callose pattern in meiocytes and megaspore tetrads is the same as in the F₁ and the parentOe. hookeri. During the development of the megaspore tetrads and the embryo sacs primary and secondary heteropolarity as well as homopolarity is observed. Estimates for the initial frequency of homo- and heteropolar tetrads at the end of the degeneration of megaspores in the tetrads immediately before the start of embryo sac development could be calculated. The F₂-plants can be arranged in three groups, distinguished by the frequency of the two polarity types. One of these groups behaves similar to the parentOe. hookeri, the two others have more homopolar tetrads. The segregation can be interpreted as recombination of genes, which influence the development of the polarity in the ovules. This is possible by crossing-over of genes between the twoRenner-complexes of the hybrid.     

The effects of prostaglandin F2α on the fine structure of the corpus luteum of the pregnant hamster

Summary The commonest intracellular organelle characteristic of the Phylum Cnidaria or Coelenterata (Subclass Zoantharia) is the spirocyst. Based on scanning and transmission electron microscopy of the tentacles of sea anemones and corals, it appears that the tip of the spirocyst is either exposed to the environment or covered by a thin plasma membrane and often has a pebbled or knobby appearance. Surrounding the spirocyst tip is a ring-like structure which seems to be formed by the junction of the enclosing cell (the spirocyte) and the tip of the spirocyst. The spirocyst thread is continuous with the capsule wall and emerges from within the apical ring during discharge. No ciliary structures appear to be associated with spirocysts. Instead, two different types of microvilli have been found: short microvilli on the spirocyte itself and long microvilli furnished by the cell or cells surrounding the spirocyte. The significance of these findings is discussed in relation to the reception of stimuli for spirocyst discharge.Thanks are due Dr. Cadet Hand for the use of facilities of the Bodega Marine Laboratory of the University of California and Dr. R.K. Thompson, P. Nemanic, H. Sampson, F. Doroshow, E. Chang and B. Miller for expert technical assistance. The use of the facilities of the Electron Microscope Laboratory and the Electronics Research Laboratory of the University of California and the Electron Microscope Laboratory of the Florida State University is gratefully acknowledged. Part of this work was made possible by NSF Grant # GB-40547 to the senior author.     

Inhibition of F1-ATPase Rotational Catalysis by the Carboxyl-terminal Domain of the ? Subunit

Escherichia coli ATP synthase (F₀F₁) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F₁-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F₁ rotation. J. Biol. Chem. 285, 42058–42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer¹⁰⁸ in loop 2 and ϵTyr¹¹⁴ in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F₁ rotation through interaction with other subunits.     

Validation of the determination of ΔF508 mutations of the cystic fibrosis gene in over 11000 mouthwashes

Mouthwashes can be used as a DNA resource for mutation detection and, because collection and DNA isolation is simple and cheap, they could in particular, be used for large numbers of samples. To determine the failure rate (the proportion of mouth samples in which no PCR product was obtained) and the specificity of buccal epithelial cell mutation detection in large numbers of samples, we collected mouthwashes and blood samples from 11413 blood donors and tested the mouthwashes for the F508 mutation, which has an estimated frequency of 75% among cystic fibrosis chromosomes in The Netherlands. Blood samples were tested for the F508 mutations only if the mutation was identified in the mouthwash or in the case of a failure to obtain PCR products. The sensitivity of the test was determined in mouthwashes of 75 F508 carriers known from earlier family studies. These samples were offered blindly between the mouthwashes of the blood donors. Both specificity and sensitivity of the mouthwash procedure were 100%. The overall failure rate was 5.6%. This large figure was caused mainly by insufficient rinsing of the mouth in one particular blood bank. Exclusion of the results of this blood bank reduced the failure rate to 1.8%. Our results also confirm that for a large number of samples the mouthwash procedure is suitable for mutation detection and, with proper instructions, can be used in community screening.     

Resolution of the effects induced by W?→?F substitutions on the conformation and dynamics of the amyloid-forming apomyoglobin mutant W7FW14F

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.     

The Biological Role of the ζ Subunit as Unidirectional Inhibitor of the F1FO-ATPase of Paracoccus denitrificans

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Conformational change of the α subunit of Escherichia coli F1 ATPase: ATP changes the trypsin sensitivity of the subunit

Conformational change in the α subunit of Escherichia coli proton-translocating ATPase was studied using trypsin. The subunit was cleaved with a small amount of trypsin (1 μg/mg subunit) to peptides of less than 8000 daltons. On the other hand, the subunit was cleaved to two main polypeptides (30,000 and 25,000 daltons) in the presence of sufficient ATP (1 mm-0.5 μm) to saturate the high-affinity site of the subunit. Analysis of digests of the subunit combined with fluorescent maleimide suggested that the subunit was digested in the middle of the polypeptide chain in the presence of the nucleotide. ADP and adenylyl imidodiphosphate had the same effect as ATP. These results suggest that the conformation of the subunit changed to form two trypsin-resistant domains upon binding of ATP to the high-affinity site.     

A Conformational Change of the γ Subunit Indirectly Regulates the Activity of Cyanobacterial F1-ATPase

The central shaft of the catalytic core of ATP synthase, the γ subunit consists of a coiled-coil structure of N- and C-terminal α-helices, and a globular domain. The γ subunit of cyanobacterial and chloroplast ATP synthase has a unique 30–40-amino acid insertion within the globular domain. We recently prepared the insertion-removed α₃β₃γ complex of cyanobacterial ATP synthase (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855–865). Although the insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition. We postulated that removal of the insertion affects the activity via a conformational change of two central α-helices in γ. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central α-helices to each other by way of a disulfide bond formation. The mutant obtained showed a significant change in ATP hydrolysis activity caused by this restriction. The highly active locked complex was insensitive to N-dimethyldodecylamine-N-oxide, suggesting that the complex is resistant to ADP inhibition. In addition, the lock affected ϵ inhibition. In contrast, the change in activity caused by removal of the γ insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the γ subunit indirectly regulates complex activity by changing both ADP inhibition and ϵ inhibition.     

Effects of the ΔF508 mutation on the structure,function, and folding of the first nucleotide-binding domain of CFTR

The fatal autosomal recessive disease cystic fibrosis (CF) is caused by mutations in the gene which encodes the cystic fibrosis transmembrane conductance regulator (CFTR). Many of these disease-causing mutations, including the deletion of F508 (F508) which accounts for approximately 70% of the disease alleles, occur in one of the two consensus nucleotide binding sequences. Peptide studies have directly demonstrated that the N-terminal nucleotide binding sequences bind adenine nucleotides. Structurally, circular dichroism spectropolarimetry indicates that this region of CFTR assumes a -stranded structure in solution. The F508 mutation causes a diminution in the amount of -stranded structure and a concomitant increase in the amount of random coil structure present, indicating that either the mutant peptide has a different native structure or that the conformational equilibrium is shifted toward a more disordered form. Furthermore, the mutant peptide is more sensitive to denaturation, indicating that F508 is a stability, or protein-folding mutant. Here we review these results and discuss their implications for interpreting the behavior of F508in situ and for the rational design of new CF drugs.     

Frequency of the cystic fibrosis ΔF508 mutation in a large sample of the French population

Summary We have determined the frequency of the cystic fibrosis (CF) ΔF508 mutation in a large sample of CF patients originating from different areas of France, including the greater Paris, Brittany, Alsace, Lorraine and Rh?ne-Alpes regions. A total of 422 CF chromosomes were studied, and the defect was found to account for 75% of the mutant alleles. In the course of the survey, a rare nucleotide sequence polymorphism leading to an isoleucine to valine substitution at position 506 of the CF transmembrane conductance regulator protein has been characterized in an unaffected individual. Our data enable the evaluation of the probabilities that a chromosome negative for the ΔF508 mutation carriers another CF defect.     

Crystal Structure of the Mg·ADP-inhibited State of the Yeast F1c10-ATP Synthase

The F₁c₁₀ subcomplex of the yeast F₁F₀-ATP synthase includes the membrane rotor part c₁₀-ring linked to a catalytic head, (αβ)₃, by a central stalk, γδϵ. The Saccharomyces cerevisiae yF₁c₁₀·ADP subcomplex was crystallized in the presence of Mg·ADP, dicyclohexylcarbodiimide (DCCD), and azide. The structure was solved by molecular replacement using a high resolution model of the yeast F₁ and a bacterial c-ring model with 10 copies of the c-subunit. The structure refined to 3.43-Å resolution displays new features compared with the original yF₁c₁₀ and with the yF₁ inhibited by adenylyl imidodiphosphate (AMP-PNP) (yF₁(I–III)). An ADP molecule was bound in both β_DP and β_TP catalytic sites. The α_DP-β_DP pair is slightly open and resembles the novel conformation identified in yF₁, whereas the α_TP-β_TP pair is very closed and resembles more a DP pair. yF₁c₁₀·ADP provides a model of a new Mg·ADP-inhibited state of the yeast F₁. As for the original yF₁ and yF₁c₁₀ structures, the foot of the central stalk is rotated by ∼40 ° with respect to bovine structures. The assembly of the F₁ central stalk with the F₀ c-ring rotor is mainly provided by electrostatic interactions. On the rotor ring, the essential cGlu⁵⁹ carboxylate group is surrounded by hydrophobic residues and is not involved in hydrogen bonding.     

The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase

The crystal structure of mitochondrial F₁-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF₁, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F₁,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F₁ and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F₁.     

The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria

The ζ subunit is a novel inhibitor of the F₁F_O-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF₁) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F₁-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599–608) by a hitherto unknown quaternary structure that was first modeled here by structural homology and protein docking. The F₁-ATPase and F₁-ζ models of P. denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F₁-ATPase at the open catalytic α_E/β_E interface, and two partial γ rotations lock the N terminus of ζ in an “inhibition-general core region,” blocking further γ rotation, while the ζ globular domain anchors it to the closed α_DP/β_DP interface. Heterologous inhibition of the F₁-ATPase of P. denitrificans by the mitochondrial IF₁ supported both the modeled ζ binding site at the α_DP/β_DP/γ interface and the endosymbiotic α-proteobacterial origin of mitochondria. In summary, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF₁ or the bacterial ϵ binds. The proposed pawl mechanism is coupled to the rotation of the central γ subunit working as a ratchet but with structural differences that make it a unique control mechanism of the nanomotor to favor the ATP synthase activity over the ATPase turnover in the α-proteobacteria.     

A study of the genus Aleuritopteris Fée in China

As a genus, Aleuritopteris was first founded by Fée upon Pteris farinosa Forsk. in 1852. The genus had been ever since, however, practically forgotten, because it did not receive a general recognition among the fern students, who considered it either as Cheilanthes (Baker. 1897, Diels, 1900, C. Christensen 1905), or placed it in Pellaea (Hooker. 1858, Hope et Wright 1903). Ching (1941) first reinstated the status of Aleuritopteris and later followed by Copeland (1947), Panigrahi (1961, 1962.) and Pichi-Sermolli (1975). In recent years, however, some pteridologists (Nayar, 1962, K. Iwatsuki and others) are still against it as a distinct genus. Our recent study of the rich material on hand has shown Aleuritopteris to be a distinct genus, according to both the morphological characteristics and geographical distribution. We further propose the possibly evolutionary relationships of Aleuritopteris with other related genera of the Cheilanthoid ferns, as indicated by the above scheme. The genus is now represcented by a little more than 30 species, of which 25 areknown in China, among which 8 species and 3 varieties are diescribed for the first time.     

Existence of Traveling Waves for the Generalized F–KPP Equation

Variation in genotypes may be responsible for differences in dispersal rates, directional biases, and growth rates of individuals. These traits may favor certain genotypes and enhance their spatiotemporal spreading into areas occupied by the less advantageous genotypes. We study how these factors influence the speed of spreading in the case of two competing genotypes under the assumption that spatial variation of the total population is small compared to the spatial variation of the frequencies of the genotypes in the population. In that case, the dynamics of the frequency of one of the genotypes is approximately described by a generalized Fisher–Kolmogorov–Petrovskii–Piskunov (F–KPP) equation. This generalized F–KPP equation with (nonlinear) frequency-dependent diffusion and advection terms admits traveling wave solutions that characterize the invasion of the dominant genotype. Our existence results generalize the classical theory for traveling waves for the F–KPP with constant coefficients. Moreover, in the particular case of the quadratic (monostable) nonlinear growth–decay rate in the generalized F–KPP we study in detail the influence of the variance in diffusion and mean displacement rates of the two genotypes on the minimal wave propagation speed.