Characterisation of subunit III and its oligomer from spinach chloroplast ATP synthase

Proton ATP synthases carry out energy conversion in mitochondria, chloroplasts, and bacteria. A key element of the membrane integral motor CFO in chloroplasts is the oligomer of subunit III: it converts the energy of a transmembrane electrochemical proton gradient into rotational movement. To enlighten prominent features of the structure-function relationship of subunit III from spinach chloroplasts, new isolation methods were established to obtain highly pure monomeric and oligomeric subunit III in milligram quantities. By Fourier-transform infrared (FTIR) and CD spectroscopy, the predominantly alpha-helical secondary structure of subunit III was demonstrated. For monomeric subunit III, a conformational change was observed when diluting the SDS-solubilized protein. Under the same conditions the conformation of the oligomer III did not change. A mass of 8003 Da for the monomeric subunit III was determined by MALDI mass spectrometry (MALDI-MS), showing that no posttranslational modifications occurred. By ionisation during MALDI-MS, the noncovalent homooligomer III14 disaggregated into its III monomers.     

Molecular dissection of the epsilon subunit of the chloroplast ATP synthase of spinach.

The gene encoding the epsilon subunit (atpE) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia coli. The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol to obtain epsilon that is as biologically active as epsilon purified from chloroplast-coupling factor 1 (CF1). Recombinant epsilon folded in this manner inhibits the ATPase activity of soluble and membrane-bound CF1 deficient in epsilon and restores proton impermeability to thylakoid membranes reconstituted with CF1 deficient in epsilon. Site-directed mutagenesis was used to generate truncations and single amino acid substitutions in the primary structure of epsilon. In the five mutants tested, alterations that weaken ATPase inhibition by recombinant epsilon affect its ability to restore proton impermeability to a similar extent, with one exception. Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeability. As in the case of E. coli, it appears that N-terminal truncations of the epsilon subunit have more profound effects than C-terminal deletions on the function of epsilon. Recombinant epsilon with six amino acids deleted from the C terminus, which is the only region of significant mismatch between the epsilon of spinach and the epsilon of Pisum sativum, inhibits ATPase activity with a reduced potency similar to that of purified pea epsilon. Four of the six amino acids are serine or threonine. These hydroxylated amino acids may be important in epsilon-CF1 interactions.     

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.     

Nucleotide binding to the isolated beta subunit of the chloroplast ATP synthase.

The beta subunit isolated from the chloroplast ATP synthase F1 (CF1) has a single dissociable nucleotide binding site, consistent with the proposed function of this subunit in nucleotide binding and catalysis. The beta subunit bound the nucleotide analogs trinitrophenyl-ATP (TNP-ATP) or trinitrophenyl-ADP (TNP-ADP) with nearly equal affinities (Kd = 1-2 microM) but did not bind trinitrophenyl-AMP. Both ATP and ADP effectively competed with TNP-ATP for binding. Other nucleoside triphosphates were also able to compete with TNP-ATP for binding to beta; their order of effectiveness (ATP greater than GTP, ITP greater than CTP) mimicked the normal substrate specificity of CF1. The single nucleotide binding site on the isolated beta subunit very closely resembles the low affinity catalytic site (site 3) of CF1 (Bruist, M.F., and Hammes, G. G. (1981) Biochemistry 20, 6298-6305), suggesting that tight nucleotide binding by other sites on the enzyme involves other CF1 subunits in addition to the beta subunit. The results are inconsistent with an earlier report (Frasch, W.D., Green, J., Caguial, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069), which suggested more than one nucleotide binding site per beta subunit. Binding of nucleotides to the isolated beta subunit was eliminated by a brief heat treatment (40 degrees C for 10 min) of the protein. A small change in the circular dichroism spectrum of beta accompanied the heat treatment indicating that a localized (rather than global) change in the folding of beta, involving at least part of the nucleotide binding domain, had occurred. Also accompanying the loss of nucleotide binding was a loss of the reconstitutive capacity of the beta subunit. ATP protected against the effects of the heat treatment.     

Structural mapping of cysteine-63 of the chloroplast ATP synthase beta subunit.

The single sulfhydryl residue (cysteine-63) of the beta subunit of the chloroplast ATP synthase F1 (CF1) was accessible to labeling reagents only after removal of the beta subunit from the enzyme complex. This suggests that cysteine-63 may be located at an interface between the beta and the alpha subunits of CF1, although alternative explanations such as a conformational change in beta brought about by its release from CF1 cannot be ruled out. Cysteine-63 was specifically labeled with [(diethylamino)methylcoumarinyl]-maleimide, and the distance between this site and trinitrophenyl-ADP at the nucleotide binding site on beta was mapped using fluorescence resonance energy transfer. Cysteine-63 is located in a hydrophobic pocket, 42 A away from the nucleotide binding site on beta.     

Recombinant production and purification of the subunit c of chloroplast ATP synthase

The unique immunoglobulin idiotype expressed on the surface of B lymphoma cells can be used as an effective antigen in tumor-specific vaccines when fused to immunostimulatory proteins and cytokines. A DNA vaccine encoding for an idiotype antibody single chain Fv (scFv) fragment fused to the Tetanus Toxin Fragment C (TTFrC) has been shown to induce protective anti-tumor responses. Protein-based strategies may be more desirable since they provide greater control over dosage, duration of exposure, and in vivo distribution of the vaccine. However, production of fusion protein vaccines containing complex disulfide bonded idiotype antibodies and antibody-derived fragments is challenging. We use an Escherichia coli-based cell-free protein synthesis platform as well as high-level expression of E. coli inclusion bodies followed by refolding for the rapid generation of an antibody fragment – TTFrC fusion protein vaccine. Vaccine proteins produced using both methods were shown to elicit anti-tumor humoral responses as well as protect from tumor challenge in an established B cell lymphoma mouse model. The development of technologies for the rapid production of effective patient-specific tumor idiotype-based fusion protein vaccines provides opportunities for clinical application.     

F1F0-ATP synthase. Binding of delta subunit to a 22-residue peptide mimicking the N-terminal region of alpha subunit

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to alpha(3)beta(3) hexagon of F(1). Proteolysis and cross-linking had suggested that N-terminal residues of alpha subunit are involved in binding delta. Here we demonstrate that a synthetic peptide consisting of the first 22 residues of alpha ("alpha N1-22") binds specifically to isolated wild-type delta subunit with high affinity (K(d) = 130 nm), accounting for a major portion of the binding energy when delta-depleted F(1) and isolated delta bind together (K(d) = 1.4 nm). Stoichiometry of binding of alpha N1-22 to delta at saturation was 1/1, showing that in intact F(1)F(0)-ATP synthase only one of the three alpha subunits is involved in delta binding. When alpha N1-22 was incubated with delta subunits containing mutations in helices 1 or 5 on the F(1)-binding face of delta, peptide binding was impaired as was binding of delta-depleted F(1). Residues alpha 6-18 are predicted to be helical, and a potential helix capping box occurs at residues alpha 3-8. Circular dichroism measurements showed that alpha N1-22 had significant helical content. Hypothetically a helical region of residues alpha N1-22 packs with helices 1 and 5 on the F(1)-binding face of delta, forming the alpha/delta interface.     

Microcrystals of tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium are catalytically active

An improved and efficient method has been developed for the purification of the tryptophan synthase alpha 2 beta 2 complex (EC 4.2.1.20) from Salmonella typhimurium containing a multicopy plasmid. Microcrystals prepared in 12% poly(ethylene glycol) 8000 containing 2.5 mM spermine are shown by scanning electron microscopy to have the same crystal habit as the larger crystals that are being used for structural analysis by X-ray crystallography. The average dimensions of the crystals are 33 microns (length) X 9 microns (width) X 3 microns (maximum thickness). Our finding that suspensions of microcrystals are active in several reactions catalyzed by the active sites of the alpha and beta 2 subunits demonstrates that both active sites are functional in the crystal and accessible to substrates. Thus the larger crystals being used for X-ray crystallographic studies should form complexes with substrates and analogues at both active sites and should yield functionally relevant structural information. A comparison of the reaction rates of suspensions of microcrystals with those of the soluble enzyme shows that the maximum rate of the crystalline enzyme is 0.8 that of the soluble enzyme in the cleavage of indole-3-glycerol phosphate (alpha reaction), 0.3 that of the soluble enzyme in the synthesis of L-tryptophan by the beta reaction or the coupled alpha beta reaction, and 2.7 that of the soluble enzyme in the serine deaminase reaction. These small differences in rates probably reflect functional differences between the crystalline and soluble enzymes since the reaction rates of the microcrystals are calculated to be virtually free of diffusional limitation under these reaction conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.     

Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium.

To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.     

A topological analysis of subunit alpha from Escherichia coli F1F0-ATP synthase predicts eight transmembrane segments

The membrane topology of subunit alpha from the Escherichia coli F1F0-ATP synthase was studied using a gene fusion technique. Fusion proteins linking different amino-terminal fragments of the alpha subunit with an enzymatically active fragment of alkaline phosphatase were constructed by both random transposition of TnphoA and site-directed mutagenesis. Those proteins with high levels of alkaline phosphatase activity are predicted to define periplasmic domains of alpha, and this was confirmed by testing for cell growth in minimal medium supplemented with polyphosphate (P greater than 75) as the sole source of phosphate. The enzymatic activity of some fusion proteins was shown to be sensitive to glucose present in the growth medium. Results from subcellular fractionation experiments suggest that these fusion proteins may be inactive even though they have a periplasmic alkaline phosphatase. The enzymatic activity appears dependent upon proteolytic release of the alkaline phosphatase moiety from its alpha subunit membrane anchor and suggests the target of glucose repression may be a protease present in the periplasm. For the topological analysis of the alpha subunit, a total of 28 unique fusion proteins were studied and the results were consistent with a model of alpha containing eight transmembrane segments, including periplasmic amino and carboxyl termini. Surprisingly, separate periplasmic domains were identified near amino acids 200, 233, and 270. These results suggest the flanking membrane spans are only 10-15 amino acids in length and not able to span a standard 30 A bilayer in an alpha-helical conformation. These short spans may have interesting mechanistic implications for the function of F0, because they contain several amino acids which appear critical for proton translocation. Finally, a fusion of alkaline phosphatase at amino acid 271, the carboxyl-terminal residue, but not at amino acid 260, was able to complement the strain RH305 (uncB-) for growth on succinate and suggests the last 11 amino acids of the alpha subunit are critical to the function of F1F0-ATP synthase.     

The catalytic role of subunit IV of the cytochrome b6-f complex from spinach chloroplast

The catalytic role of subunit IV, the Mr 17,000 protein, in the chloroplast cytochrome b6-f complex was established through trypsinolysis of the complex under controlled conditions. When purified chloroplast cytochrome b6-f complex, 1 mg/ml, in 50 mM Tris-succinate buffer (pH 7.0) containing 1% sodium cholate and 10% glycerol is treated with 80 micrograms of trypsin at room temperature for various lengths of time, the activity of the cytochrome b6-f complex decreases as the incubation time increases. A maximal inactivation of 80% is reached at 7 min of incubation. The trypsin inactivation is accompanied by the destruction of the proton translocation activity of the complex. No alteration of absorption and EPR spectral properties was observed in the trypsin-inactivated complex. Subunit IV is the only subunit in the cytochrome b6-f complex that is digested by trypsin, and the degree of digestion correlates with the decrease of electron transfer activity. The binding of azido-Q to subunit IV of the complex decreases as the extent of inactivation of the cytochrome b6-f complex by trypsin increases. The residue molecular mass of trypsin cleaved subunit IV is about 14 kDa, suggesting that the cleavage site is at lysine 119 or arginine 125 or 126. When the thylakoid membrane was assayed for cytochrome b6-f complex activity, very little activity was observed; and the activity was not sensitive to trypsinolysis. Upon sonication, activity and sensitivity to trypsinolysis was greatly increased, suggesting that subunit IV protrudes from the lumen side of the membrane.     

The tryptophan synthase alpha 2 beta 2 complex. Cleavage of a flexible loop in the alpha subunit alters allosteric properties

This study explores the catalytic and allosteric roles of a flexible loop in tryptophan synthase. Trypsin is known to cleave the tryptophan synthase alpha 2 beta 2 complex in an alpha subunit loop at Arg-188. Cleavage yields an active "nicked" alpha 2 beta 2 derivative. The new results provide evidence that the alpha subunit loop serves two important roles: substrate binding and communicating the effects of substrate binding to the beta subunit. A role for the loop in substrate binding is supported by our finding that addition of a substrate analogue of the alpha subunit, alpha-glycerol 3-phosphate, decreases the rate of cleavage by trypsin. An allosteric role for the loop is supported by the finding although the native alpha 2 beta 2 complex is strongly inhibited by alpha-glycerol 3-phosphate, the nicked alpha 2 beta 2 complex is desensitized to this inhibition. The time course of proteolysis in the presence and absence of alpha-glycerol 3-phosphate is followed by sodium dodecyl sulfate-gel electrophoresis and by assays of activity in the presence and absence of alpha-glycerol 3-phosphate. We use spectroscopic measurements of the pyridoxal phosphate-L-tryptophan intermediates at the active site of the beta subunit to determine the affinity of the native and nicked enzymes for L-tryptophan and alpha-glycerol 3-phosphate. Although cleavage alters the equilibrium distribution of intermediates and reduces the affinity for alpha-glycerol 3-phosphate, it has little effect on the affinity for amino acids bound to the beta subunit. We conclude that the loop in the alpha subunit is important for ligand binding and for communicating the effects of ligand binding from the alpha subunit to the beta subunit in the alpha 2 beta 2 complex.     

Selective modification of an alpha subunit of chloroplast coupling factor 1

Lucifer yellow (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulf onate), a fluorescent probe that can react covalently with sulfhydryl or amino groups, has been used to modify chloroplast coupling factor 1 (CF1). Conditions are described under which Lucifer yellow selectively labels the alpha subunit of CF1 to the extent of about 1 mol of probe per mole of CF1. An especially reactive amino group is apparently labeled, and modification has little effect on the ATPase activity of the enzyme. Lucifer yellow is a useful probe for fluorescence energy transfer measurements. The distances between this probe and fluorescent and absorbing molecules attached to seven specific sites on the beta, gamma, and epsilon subunits were determined. These distances converge to a single location. In addition to providing further information about the structure of CF1, these results suggest that the alpha subunits of CF1 are not structurally equivalent.     

The 55-kDa polypeptide released from spinach thylakoid membranes with 1 M LiCl is not the beta subunit of chloroplast F1

It was reported by Frasch et al. (Frasch, W. D., Green, J., Caguiat, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069) that washing spinach thylakoid membranes with 1 M LiCl caused the release of the beta subunit of chloroplast F1 (CF1) which, existing as 180-kDa complexes of beta 3, retained considerable ATPase activity. We repeated their procedures and confirmed that a CF1 beta-like 55-kDa polypeptide was a major constituent of the 1 M LiCl-washed extract. However, the extract contained another polypeptide of which the Mr was 14,000, and these two polypeptides comprised a complex with approximate Mr 550,000 that had the same mobility in native polyacrylamide gel electrophoresis as that of ribulose-1,5-bisphosphate carboxylase. Only very low ATPase activity, less than 1% of the reported value, was detected for the extract and the purified complex. Antibody against the beta subunit of F1 from a thermophilic bacterium PS3 showed a clear cross-reactivity with the CF1 beta subunit but not with the 55-kDa polypeptide. Analysis of the N-terminal amino acid sequences of the 55- and 14-kDa polypeptides and the whole complex revealed that the complex was ribulose-1,5-bisphosphate carboxylase and that the 55- and 14-kDa polypeptides were its large and small subunits, respectively.     

Molecular devices of chloroplast F(1)-ATP synthase for the regulation

In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential difference of protons (Delta(micro)H(+)) is small, e.g. at low light intensity or in the dark. To prevent this wasteful consumption of ATP, the activity of chloroplast ATP synthase is regulated as the occasion may demand. As regulation systems Delta(micro)H(+) activation, thiol modulation, tight binding of ADP and the role of the intrinsic inhibitory subunit epsilon is well documented. In this article, we discuss recent progress in understanding of the regulation system of the chloroplast ATP synthase at the molecular level.     

Mechanism of release of active alpha subunit from dimeric alpha beta avian myeloblastosis virus DNA polymerase.

Storage of the dimeric (alphabeta) form of avian myeloblastosis virus (AMV) DNA polymerase in glycerol resulted in the release of the smaller alpha subunit, as detected by glycerol gradient sedimentation. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme stored in glycerol showed the concomitant appearance of several polypeptides and a lowering in the level of both beta and alpha components. This reduction appears to be the result of cleavages introduced by traces of hydrolytic activity present in glycerol samples. An enhancement of alpha subunit released, as detected by activity profile, was also achieved upon direct but limited exposure of purified avian myeloblastosis virus DNA polymerase to carboxymethyl-cellulose-bound trypsin matrix. Electrophoretic analysis of digested enzyme revealed a progressive fragmentation, with simultaneous increase in the alpha subunit and decrease in the beta subunit.     

Structure and function of the tryptophan synthase alpha(2)beta(2) complex. Roles of beta subunit histidine 86

To probe the structural and functional roles of active-site residues in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium, we have determined the effects of mutation of His(86) in the beta subunit. His(86) is located adjacent to beta subunit Lys(87), which forms an internal aldimine with the pyridoxal phosphate and catalyzes the abstraction of the alpha-proton of L-serine. The replacement of His(86) by leucine (H86L) weakened pyridoxal phosphate binding approximately 20-fold and abolished the circular dichroism signals of the bound coenzyme and of a reaction intermediate. Correlation of these results with previous crystal structures indicates that beta-His(86) plays a structural role in binding pyridoxal phosphate and in stabilizing the correct orientation of pyridoxal phosphate in the active site of the beta subunit. The H86L mutation also altered the pH profiles of absorbance and fluorescence signals and shifted the pH optimum for the synthesis of L-tryptophan from pH 7.5 to 8.8. We propose that the interaction of His(86) with the phosphate of pyridoxal phosphate and with Lys(87) lowers the pK(a) of Lys(87) in the wild-type alpha(2)beta(2) complex and thereby facilitates catalysis by Lys(87) in the physiological pH range.     

Large-scale purification of active cytochrome b6/f complex from spinach chloroplasts

A preparation is described through which large quantities of pure, active cytochrome b6/f complex can be isolated from spinach chloroplasts. The resulting complex is at least 90% pure with respect to the maximum content of redox centers, consists of four polypeptides according to polyacrylamide gel electrophoresis, and lacks both ferredoxin: NADP+ oxidoreductase and the high molecular weight form of cytochrome f seen in some other preparations. The complex contains 2 mol b6 and 2 atoms of nonheme iron per mole of cytochrome f, and possesses a high plastoquinol-plastocyanin oxidoreductase activity (Cyt f turnover no. 20-35 s-1). The present preparation should be helpful in the effort to crystallize the cytochrome b6/f complex.     

ATP hydrolysis catalyzed by a beta subunit preparation purified from the chloroplast energy transducing complex CF1.CF0

Washing thylakoid membranes with 1 M LiCl causes the release of the beta subunit from the chloroplast energy transducing complex (CF1.CF0) in spinach chloroplasts. This protein purifies by size exclusion chromatography as a 180-kDa aggregate and, thus, is probably composed of a trimer of beta polypeptides. The purified aggregate binds ADP to a high and a low affinity site with dissociation constants of 15 and 202 microM, respectively. Mg2+ is required for ADP to bind to both sites. Manganese binds to the protein in a cooperative manner to at least two sites with high affinity. The beta subunit preparation catalyzes Mg2+-dependent ATP hydrolysis at rates which are comparable to other subunit-deficient CF1 preparations and is increased by treatments known to activate the Mg2+-ATPase activity of CF1. However, Ca2+ is not an effective cofactor for this reaction and treatments which activate the Ca2+-ATPase of CF1 are either ineffective or inhibitory.