A putative alpha-helical porin from Corynebacterium glutamicum

The cell wall of Corynebacterium glutamicum contains a mycolic acid layer, which is a protective nonpolar barrier similar to the outer membrane of Gram-negative bacteria. The exchange of material across this barrier requires porins. Porin B (PorB) is one of them. Recombinant PorB has been produced in Escherichia coli, purified, crystallized and analyzed by X-ray diffraction, yielding 16 independent molecular structures in four different crystal forms at resolutions up to 1.8 Å. All 16 molecules have the same globular core, which consists of 70 residues forming four α-helices tied together by a disulfide bridge. The 16 structures vary greatly with respect to the 29 residues in the N- and C-terminal extensions. Since corynebacteria belong to the group of mycolata that includes some prominent human pathogens, the observed structure may be of medical relevance. Due to the clearly established solid structure of the core, the native porin has to be oligomeric, and the reported structure is one of the subunits. An α-helical porin in a bacterial outer envelope is surprising because all presently known structures of such porins consist of β-barrels. Since none of the four crystal packing arrangements was compatible with an oligomeric membrane channel, we constructed a model of such an oligomer that was consistent with all available data of native PorB. The proposed model is based on the required polar interior and nonpolar exterior of the porin, on a recurring crystal packing contact around a 2-fold axis, on the assumption of a simple C_n symmetry (a symmetric arrangement around an n-fold axis), on the experimentally established electric conductivity and anion selectivity and on the generally observed shape of porin channels.     

Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp.

Mitochondrial F₁F₀-ATP synthase of chlorophycean algae is a dimeric complex of 1600 kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp.     

Clustering of water bodies in unpolluted and polluted environments based on Escherichia coli phylogroup abundance using a simple interaction database

Different types of water bodies, including lakes, streams, and coastal marine waters, are often susceptible to fecal contamination from a range of point and nonpoint sources, and have been evaluated using fecal indicator microorganisms. The most commonly used fecal indicator is Escherichia coli, but traditional cultivation methods do not allow discrimination of the source of pollution. The use of triplex PCR offers an approach that is fast and inexpensive, and here enabled the identification of phylogroups. The phylogenetic distribution of E. coli subgroups isolated from water samples revealed higher frequencies of subgroups A₁ and B2₃ in rivers impacted by human pollution sources, while subgroups D₁ and D₂ were associated with pristine sites, and subgroup B1 with domesticated animal sources, suggesting their use as a first screening for pollution source identification. A simple classification is also proposed based on phylogenetic subgroup distribution using the w-clique metric, enabling differentiation of polluted and unpolluted sites.     

Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a “pearl-chain” of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O₂ molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 Å cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 Å resolution (FSC_1/2-bit criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 Å structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

The flagellar filament structure of the extreme acidothermophile Sulfolobus shibatae B12 suggests that archaeabacterial flagella have a unique and common symmetry and design

Archaea, constituting a third domain of life between Eubacteria and Eukarya, characteristically inhabit extreme environments. They swim by rotating flagellar filaments that are phenomenologically and functionally similar to those of eubacteria. However, biochemical, genetic and structural evidence has pointed to significant differences but even greater similarity to eubacterial type IV pili. Here we determined the three-dimensional symmetry and structure of the flagellar filament of the acidothermophilic archaeabacterium Sulfolobus shibatae B12 using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Processing of the cryo-negatively stained filaments included analysis of their helical symmetry and subsequent single particle reconstruction. Two filament subunit packing arrangements were identified: one has helical symmetry, similar to that of the extreme halophile Halobacterium salinarum, with ten subunits per 5.3 nm repeat and the other has helically arranged stacked disks with C₃ symmetry and 12 subunits per repeat. The two structures are related by a slight twist. The S. shibatae filament has a larger diameter compared to that of H. salinarum, at the opposite end of the archaeabacterial phylogenetic spectrum, but the basic three-start symmetry and the size and arrangement of the core domain are conserved and the filament lacks a central channel. This similarity suggests a unique and common underlying symmetry for archaeabacterial flagellar filaments.     

In glucose-limited continuous culture the minimum substrate concentration for growth,smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii,an environmentally abundant bacterium

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters μ_max and K_s were determined for growth with glucose. For both strains, μ_max was determined in batch culture after different precultivation conditions. In the case of C. heintzii, μ_max was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h⁻¹ C. heintzii grew immediately with a μ_max of 0.17±0.03 h⁻¹. After five transfers in batch culture, μ_max had increased only slightly to 0.18±0.03 h⁻¹. A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D=0.075 h⁻¹ into glucose-excess batch medium E. coli grew only after an acceleration phase of ∼3.5 h with a μ_max of 0.52 h⁻¹. After 120 generations and several transfers into fresh medium, μ_max had increased to 0.80±0.03 h⁻¹. For long-term adapted chemostat-cultivated cells, a K_s for glucose of 15 μg l⁻¹ for C. heintzii, and of 35 μg l⁻¹ for E. coli, respectively, was determined in ¹⁴C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h⁻¹. Using the determined pure culture parameter values for K_s and μ_max, it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s_min). The values estimated for s_min were dependent on growth rate; at D=0.05 h⁻¹, it was 12.6 and 0 μg l⁻¹ for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h⁻¹, s_min for E. coli had to be raised to 34.9 μg l⁻¹ whereas s_min for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K_s value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s_min.     

The First Three-Dimensional Structure of Phosphofructokinase from Saccharomyces cerevisiae Determined by Electron Microscopy of Single Particles

Phosphofructokinaseis a key regulatory enzyme of the glycolytic pathway. We have determined the structure of this enzyme from Saccharomyces cerevisiae to a resolution of 2.0 nm. This is the first structure available for this family of enzymes in eukaryotic organisms. Phosphofructokinase is an octamer composed of 4α and 4β subunits arranged in a dihedral point group symmetry D₂. The enzyme has a very open and elongated structure, with dimensions of 24 nm in length and 17 nm in width. The final structure, calculated from 0° tilt projections of the molecule at random orientations using as reference the volume obtained by the random conical reconstruction technique in ice, has allowed us to discern the shapes of the subunits and their mutual arrangement in the octamer.     

10-Å CryoEM Structure and Molecular Model of the Myriapod (Scutigera) 6 × 6mer Hemocyanin: Understanding a Giant Oxygen Transport Protein

Oxygen transport in Myriapoda is maintained by a unique 6 × 6mer hemocyanin, that is, 36 subunits arranged as six hexamers (1 × 6mers). In the sluggish diplopod Spirostreptus, the 1 × 6mers seem to operate as almost or fully independent allosteric units (h ∼ 1.3; P₅₀ ∼ 5 torr), whereas in the swift centipede Scutigera, they intensively cooperate allosterically (h ∼ 10; P₅₀ ∼ 50 torr). Here, we show the chemomechanical basis of this differential behavior as deduced from hybrid 6 × 6mer structures, obtained by single-particle cryo-electron microscopy of the Scutigera 6 × 6mer (10.0 Å resolution according to the 0.5 criterion) and docking of homology-modeled subunits from Scutigera and two diplopods, Spirostreptus and Polydesmus. The Scutigera 6 × 6mer hemocyanin is a trigonal antiprism assembled from six smaller trigonal antiprisms (1 × 6mers), thereby exhibiting D3 point group symmetry. It can be described as two staggered 3 × 6mers or three oblique 2 × 6mers. Topologically, the 6 × 6mer is subdivided into six subunit zones, thereby exhibiting a mantle (24 subunits) and a core (12 subunits). The six hexamers are linked by 21 bridges, subdivided into five types: two within each 3 × 6mer and three between both 3 × 6mers. The molecular models of the 6 × 6mer reveal intriguing amino acid appositions at these inter-hexamer interfaces. Besides opportunities for salt bridges, we found pairs of carboxylate residues for possible bridging via a Ca²⁺ or Mg²⁺ ion. Moreover, we detected histidine clusters, notably in Scutigera, allowing us to advance hypotheses as to how the hexamers are allosterically coupled in centipede hemocyanin and why they act more independently in diplopod hemocyanin.     

Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths

A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5–6 months old Assaf lambs using Al(OH)₃ or Escherichia coli lipopolysaccharide + inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%–85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

13N as a tracer for studying glutamate metabolism

This mini-review summarizes studies my associates and I carried out that are relevant to the topic of the present volume [i.e. glutamate dehydrogenase (GDH)] using radioactive ¹³N (t_1/2 9.96 min) as a biological tracer. These studies revealed the previously unrecognized rapidity with which nitrogen is exchanged among certain metabolites in vivo. For example, our work demonstrated that (a) the t_1/2 for conversion of portal vein ammonia to urea in the rat liver is ∼10-11 s, despite the need for five enzyme-catalyzed steps and two mitochondrial transport steps, (b) the residence time for ammonia in the blood of anesthetized rats is ≤7-8 s, (c) the t_1/2 for incorporation of blood-borne ammonia into glutamine in the normal rat brain is <3 s, and (d) equilibration between glutamate and aspartate nitrogen in rat liver is extremely rapid (seconds), a reflection of the fact that the components of the hepatic aspartate aminotransferase reaction are in thermodynamic equilibrium. Our work emphasizes the importance of the GDH reaction in rat liver as a conduit for dissimilating or assimilating ammonia as needed. In contrast, our work shows that the GDH reaction in rat brain appears to operate mostly in the direction of ammonia production (dissimilation). The importance of the GDH reaction as an endogenous source of ammonia in the brain and the relation of GDH to the brain glutamine cycle is discussed. Finally, our work integrates with the increasing use of positron emission tomography (PET) and nuclear magnetic resonance (NMR) to study brain ammonia uptake and brain glutamine, respectively, in normal individuals and in patients with liver disease or other diseases associated with hyperammonemia.     

Octameric structure of the human bifunctional enzyme PAICS in purine biosynthesis

Phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is an important bifunctional enzyme in de novo purine biosynthesis in vertebrate with both 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide synthetase (SAICARs) activities. It becomes an attractive target for rational anticancer drug design, since rapidly dividing cancer cells rely heavily on the purine de novo pathway for synthesis of adenine and guanine, whereas normal cells favor the salvage pathway. Here, we report the crystal structure of human PAICS, the first in the entire PAICS family, at 2.8 Å resolution. It revealed that eight PAICS subunits, each composed of distinct AIRc and SAICARs domains, assemble a compact homo-octamer with an octameric-carboxylase core and four symmetric periphery dimers formed by synthetase domains. Based on structural comparison and functional complementation analyses, the active sites of SAICARs and AIRc were identified, including a putative substrate CO₂-binding site. Furthermore, four symmetry-related, separate tunnel systems in the PAICS octamer were found that connect the active sites of AIRc and SAICARs. This study illustrated the octameric nature of the bifunctional enzyme. Each carboxylase active site is formed by structural elements from three AIRc domains, demonstrating that the octamer structure is essential for the carboxylation activity. Furthermore, the existence of the tunnel system implies a mechanism of intermediate channeling and suggests that the quaternary structure arrangement is crucial for effectively executing the sequential reactions. In addition, this study provides essential structural information for designing PAICS-specific inhibitors for use in cancer chemotherapy.     

Oligomerization of the SPP1 scaffolding protein

Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His₆) and purified. The protein is an α-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His₆ is structurally a dimer of dimers. Incubation at temperatures above 37 °C correlated with a reduction of its α-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 °C. Refolding of gp11-His₆ thermally denatured at 65 °C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (∼ 90%) and a smaller fraction of 2.4 S dimers (∼ 10%). This population of gp11-His₆ was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His₆ showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His₆.     

Diplogasteroides asiaticus n. sp. is Associated with Monochamus alternatus in Japan

Diplogasteroides asiaticus n. sp. is described and illustrated, and its molecular profile and phylogenetic status within the family Diplogastridae are inferred. Morphologically, the new species is characterized by its stomatal structure, a tube-like stoma with three small, rod-like dorsal teeth and two subventral ridges; a spicule clearly ventrally bent at 1/3 from the anterior end; a gubernaculum with a rounded anterior end and sharply pointed distal end in lateral view; nine pairs of genital papillae with an arrangement of <v1, (v2, v3d), C, v4, ad, ph, (v5, v6, v7), pd>; a short tail spike in males; and a well-developed receptaculum seminis, i.e., the antiparallel blind sacs of the uteri beyond the vulva region and elongated conical tail in females. This new species is morphologically similar to D. haslacheri, but it can be distinguished by the morphology of the somewhat shorter tail in females. D. asiaticus n. sp. shares high sequence conservation with D. andrassyi as there is only one base pair difference in the nearly full-length 18S rDNA and seven base pair differences in the D2-D3 expansion segments of the 28S rDNA. Despite this sequence conservation, the species status of D. asiaticus n. sp. was confirmed using the biological species concept, as D. asiaticus n. sp. and D. andrassyi failed to generate viable F₂ progeny in hybridization tests.     

Repeated Sampling to Determine the Precision of Estimating Nematode Population Densities

The first phase of this study involved repeated samplings of five fields using composite samples of 10, 20, 40, and 80 soil cores, to determine the precision of nematode assays. The second phase focused on randomly selecting two and four 2-ha subunits (data on Meloidogyne spp.) of 24 fields ranging from 6 to 40 ha and computing the precision of estimated means for these numbers ofsubunits versus the general field mean (based on all 2-ha subunits). Average numbers of nematodes from most samples containing Meloidogyne spp., Heterodera glycines, Helicotylenchus dihystera, Scutellonema brachyurum, and (or) Hoplolaimus galeatus were within 50% of the overall means. Coefficient of variation (CV) values were generally lower for 40 cores than for 10, 20, and 80 cores per sample. When data for all nematodes and fields were combined, this value was lowest for 40 and 80 cores. The CV values were higher for Meloidogyne spp. than for H. glycines. Means of two samplings increased the probability of obtaining numbers nearer the mean for that field than numbers from a single composite sample. For the second phase, population estimates of Meloidogyne spp. based on four 2-ha subunits generally were closer to field means than were those for two subunits. Sampling precision with these subunits diminished greatly in large fields with variable soils and (or) mixed cropping histories. Either two or four subunits gave population estimates within 3-20% of the field mean in most instances. The mean man hours required for sampling ca. 2-ha parcels of 4-20-ha fields was 0.54 hours.     

Structural model of the transmembrane Fo rotary sector of H-transporting ATP synthase derived by solution NMR and intersubunit cross-linking in situ

H⁺-transporting, F₁F_o-type ATP synthases utilize a transmembrane H⁺ potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating β subunits of the extramembranous F₁ sector of the enzyme, synthesis being driven by rotation of the γ subunit in the center of the F₁ molecule between the alternating catalytic sites . The H⁺ electrochemical potential is thought to drive γ subunit rotation by first coupling H⁺ transport to rotation of an oligomeric rotor of c subunits within the transmembrane F_o sector. The γ subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the γ and ε subunits of F₁. In this essay we will review recent studies on the Escherichia coli F_o sector. The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp⁶¹ centered in the second transmembrane helix (TMH). A model for the structural organization of the c₁₀ oligomer in F_o was deduced from extensive cross-linking studies and by molecular modeling. The model indicates that the H⁺-carrying carboxyl of subunit c is occluded between neighboring subunits of the c₁₀ oligomer and that two c subunits pack in a “front-to-back” manner to form the H⁺ (cation) binding site. In order for protons to gain access to Asp⁶¹ during the protonation/deprotonation cycle, we propose that the outer, Asp⁶¹-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp⁶¹ protonation/deprotonation drives the rotation of the c-ring. The NMR structures of wild-type subunit c differs according to the protonation state of Asp⁶¹. The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional mutant proteins in which critical residues have been switched between TMH-1 and TMH-2. The structural information is considered in the context of the possible mechanism of rotary movement of the c₁₀ oligomer during coupled synthesis of ATP.