RESUMO
The P-type ATPases are integral membrane proteins that generate essential transmembrane ion gradients in virtually all living cells. The structures of two of these have recently been elucidated at a resolution of 8 A. When considered together with the large body of biochemical information that has accrued for these transporters and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by these proteins.
Assuntos
ATPases Transportadoras de Cálcio/fisiologia , ATPases Translocadoras de Prótons/fisiologia , Animais , Transporte Biológico , ATPases Transportadoras de Cálcio/química , Citoplasma , Conformação Proteica , ATPases Translocadoras de Prótons/química , Relação Estrutura-AtividadeRESUMO
Electron cryocrystallography of precipitant-induced two-dimensional surface crystals of the neurospora plasma membrane H+ - ATPase and tubular crystals of the sarcoplasmic reticulum Ca(2+)-ATPase has recently yielded structure maps for these ion transporters at a resolution of about 8 A. The membrane-embedded regions of these closely related enzymes are similar, but the cytoplasmic regions appear to be significantly different.
Assuntos
ATPases Transportadoras de Cálcio/química , ATPases Translocadoras de Prótons/química , ATPases Transportadoras de Cálcio/metabolismo , Modelos Moleculares , Conformação Proteica , ATPases Translocadoras de Prótons/metabolismoRESUMO
The Neurospora crassa plasma membrane H+-ATPase is rapidly inactivated in the presence of diethyl pyrocarbonate (DEP). The reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 M-1.min-1 at pH 6.9 and 25 degrees C. The difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of N-carbethoxyhistidine. No change in the absorbance of the inhibited ATPase at 278 nm or in the number of modifiable sulfhydryl groups is observed, indicating that the inhibition is not due to tyrosine or cysteine modification, and the inhibition is irreversible, ruling out serine residues. Furthermore, pretreatment of the ATPase with pyridoxal phosphate/NaBH4 under the conditions of the DEP treatment does not inhibit the ATPase and does not alter the DEP inhibition kinetics, indicating that the inactivation by DEP is not due to amino group modification. The pH dependence of the inactivation reaction indicates that the essential residue has a pKa near 7.5, and the activity lost as a result of H+-ATPase modification by DEP is partially recovered after hydroxylamine treatment at 4 degrees C. Taken together, these results strongly indicate that the inactivation of the H+-ATPase by DEP involves histidine modification. Analyses of the inhibition kinetics and the stoichiometry of modification indicate that among eight histidines modified per enzyme molecule, only one is essential for H+-ATPase activity. Finally, ADP protects against inactivation by DEP, indicating that the essential residue modified may be located at or near the nucleotide binding site.
Assuntos
Dietil Pirocarbonato/farmacologia , Formiatos/farmacologia , Histidina/análise , Neurospora crassa/metabolismo , Neurospora/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Sítios de Ligação , Catálise , Membrana Celular/enzimologia , Concentração de Íons de Hidrogênio , Hidroxilamina , Hidroxilaminas/farmacologia , Neurospora crassa/enzimologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Espectrofotometria Ultravioleta , Compostos de Sulfidrila/análiseRESUMO
A system for expression and facile purification of the human P-glycoprotein (Pgp) from the yeast Saccharomyces cerevisiae is described. The wild-type human mdr1 cDNA was cloned into a high copy number yeast expression vector under the control of the constitutive promoter of the yeast plasma membrane H+-ATPase. Western blots of membranes from the stable transformants confirmed that the Pgp is expressed in yeast cells in amounts approximately 0.4% of the total yeast membrane protein. Density gradient sedimentation analysis of the yeast membranes indicated that the expressed Pgp is localized in the plasma membrane. Yeast cells transformed with the Pgp expression plasmid acquire increased resistance to valinomycin, suggesting that the expressed Pgp is properly folded and functional. The expressed Pgp can be solubilized from the yeast membranes with lysophosphatidylcholine, and when tagged with ten histidines at its C-terminus, can be readily purified to about 90% homogeneity by Ni2+ affinity chromatography. About 50 microg of the Pgp can be purified from 20 mg of crude yeast membranes. The purified human Pgp exhibits a verapamil-stimulated ATPase activity and the maximal activity is 2.5 +/- 0.5 micromol/min per mg of Pgp, suggesting that the purified Pgp from yeast is highly functional. The Pgp expressed in yeast has the same electrophoretic mobility (ca. 130 kDa) as the Pgp produced in Sf9 insect cells and is unaffected by N-glycosidase treatment, suggesting that it is not glycosylated. Because of the relative ease of growing yeast in massive quantities this expression system appears to be excellent for producing this membrane transporter at levels sufficient for further biochemical and biophysical studies, and for site-directed mutagenesis studies as well.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar/biossíntese , Humanos , Plasmídeos , Saccharomyces cerevisiae/genéticaRESUMO
The topographic location of the region comprising amino acids 359-440 of the Neurospora crassa plasma membrane H(+)-ATPase has been elucidated using reconstituted proteoliposomes and protein chemical techniques. Proteoliposomes containing H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were cleaved with trypsin and the resulting digest was subjected to centrifugation on a glycerol step gradient to separate the released and liposome-bound peptides. The released peptides were recovered in the upper regions of the step gradient, whereas the liposome-bound peptides were recovered near the 40% glycerol interface. The released peptides present in the upper fractions were reduced, 14C-carboxy-methylated, and then separated by high performance liquid chromatography. Two radioactive cysteine-containing peptides with retention times of about 162 and 182 min were identified as H(+)-ATPase peptides comprising residues Leu363-Lys379 and Leu388-Arg414, respectively, by comparison to standards prepared from the purified ATPase. This information thus establishes a cytoplasmic location for residues 359-418 in the H(+)-ATPase polypeptide chain. It also infers a cytoplasmic location for residues 419-440, since this stretch of amino acids is too short to cross the membrane and return between regions known to be cytoplasmically located. These results and the results of other recent experiments establish the topographical location of nearly all of the 919 residues in the H(+)-ATPase molecule.
Assuntos
Aminoácidos/análise , Membrana Celular/enzimologia , Citoplasma/enzimologia , Neurospora crassa/enzimologia , ATPases Translocadoras de Prótons/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Oxirredução , ATPases Translocadoras de Prótons/isolamento & purificação , TripsinaRESUMO
Recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) has been produced in a Saccharomyces cerevisiae expression system used previously to produce transport ATPases with high yields. The arrangement of the bases in the region immediately upstream from the ATG start codon of the CFTR is extremely important for high expression levels. The maximal CFTR expression level is about 5-10% of that in Sf9 insect cells as judged by comparison of immunoblots. Upon sucrose gradient centrifugation, the majority of the CFTR is found in a light vesicle fraction separated from the yeast plasma membrane in a heavier fraction. It thus appears that most of expressed CFTR is not directed to the plasma membrane in this system. CFTR expressed in yeast has the same mobility (ca. 140 kDa) as recombinant CFTR produced in Sf9 cells in a high resolution SDS-PAGE gel before and after N-glycosidase F treatment, suggesting that it is not glycosylated. The channel function of the expressed CFTR was measured by an isotope flux assay in isolated yeast membrane vesicles and single channel recording following reconstitution into planar lipid bilayers. In the isotope flux assay, protein kinase A (PKA) increased the rate of 125I- uptake by about 30% in membrane vesicles containing the CFTR, but not in control membranes. The single channel recordings showed that a PKA-activated small conductance anion channel (8 pS) with a linear I-V relationship was present in the CFTR membranes, but not in control membranes. These results show that the human CFTR has been expressed in functional form in yeast. With the reasonably high yield and the ability to grow massive quantities of yeast at low cost, this CFTR expression system may provide a valuable new source of starting material for purification of large quantities of the CFTR for biochemical studies.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Expressão Gênica , Saccharomyces cerevisiae/genética , Animais , Membrana Celular/metabolismo , Centrifugação com Gradiente de Concentração , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Humanos , Mutagênese Sítio-Dirigida , Plasmídeos , Proteínas Recombinantes , Saccharomyces cerevisiae/ultraestrutura , Spodoptera/metabolismoRESUMO
The purified H(+)-ATPase of the Neurospora crassa plasma membrane has been reconstituted by a gel filtration method into lipidic vesicles using sodium deoxycholate as the detergent. Reconstitution was performed for lipid/ATPase ratios ranging from 1000:1 to 5:1 (w/w). Whatever the lipid/ATPase ratio, the ATPase molecules completely associate with the lipid vesicles. The ATPase specific activity is identical for all proteoliposomes regardless of the lipid/ATPase ratio, but the H+ transport decreases at high protein/lipid ratios, suggesting that the proteoliposomes are more leaky to H+ as the amount of protein inserted into the lipidic membrane increases. Analysis of the fragments generated by trypsin proteolysis in the presence and in the absence of MgATP+ vanadate indicate that most of the reconstituted ATPase molecules are able to assume the transition state of the enzyme dephosphorylation reaction, and are therefore functional. The orientation (inside-out or rightside-out) of the ATPase molecules in the vesicles is independent of the lipid/ATPase ratio chosen for the reconstitution. For all the lipid/ATPase ratios tested, most of the ATPase molecules (> 99%) expose their cytoplasmic side to the outside of the reconstituted proteoliposomes. The size of the vesicles increases parallel to the ATPase amount. Although the H+ leakiness of our preparation at low lipid/protein ratios prevents proton pumping measurements, the reconstitution procedure described here has the main advantage on other procedures to allow the obtention of vesicles at high protein-to-lipid ratios, facilitating further structural characterization of the ATPase by biochemical and biophysical techniques. Therefore, the procedure described here could be of general interest in the field of membrane protein study.
Assuntos
Neurospora crassa/enzimologia , ATPases Translocadoras de Prótons/isolamento & purificação , Membrana Celular/enzimologia , Cromatografia em Gel/métodos , Detergentes , Ativação Enzimática , Lipossomos/síntese química , Neurospora crassa/ultraestrutura , Proteolipídeos/síntese química , ATPases Translocadoras de Prótons/metabolismo , TripsinaRESUMO
Large two-dimensional crystals of H+-ATPase, a 100 kDa integral membrane protein, were grown directly onto the carbon surface of an electron microscope grid. This procedure prevented the fragmentation that is normally observed upon transfer of the crystals from the air-water interface to a continuous carbon support film. Crystals grown by this method measure approximately 5 microm across and have a thickness of approximately 240 A. They are of better quality than the monolayers previously obtained at the air-water interface, yielding structure factors to at least 8 A in-plane resolution by electron image processing. Unlike most other two-dimensional crystals of membrane proteins they do not contain a lipid bilayer, but consist of detergent-protein micelles of H+-ATPase hexamers tightly packed on a trigonal lattice. The crystals belong to the two-sided plane group p321 (a=b=165 A), containing two layers of hexamers related by an in-plane axis of 2-fold symmetry. The protein is in contact with the carbon surface through its large, hydrophilic 70 kDa cytoplasmic portion, yet due to the presence of detergent in the crystallizing buffer, the hydrophobicity of the carbon surface does not appear to affect crystal formation. Surface crystallisation may be a useful method for other proteins which form fragile two-dimensional crystals, in particular if conditions for obtaining three-dimensional crystals are known, but their quality or stability is insufficient for X-ray structure determination.
Assuntos
Membrana Celular/enzimologia , Cristalografia/métodos , ATPases Translocadoras de Prótons/química , Carbono , Cristalização , Elétrons , Aumento da Imagem , Microscopia Eletrônica , Neurospora crassa/enzimologia , Conformação Proteica , Propriedades de SuperfícieRESUMO
Multidrug-resistant (MDR) tumor cells reduce the toxicity of antineoplastic drugs by an energy-dependent active efflux mechanism mediated by the MDR1 gene product, the P-glycoprotein (Pgp). Pgp expressed in cultured Sf9 insect cells has been shown to exhibit a high capacity ATPase activity in the presence of a variety of drugs known to be transported by the Pgp (Sarkadi et al., J Biol Chem 267: 4854-4858, 1992). The strict dependence of the Pgp ATPase activity on the presence of transport substrates indicates that the drug-stimulated ATPase activity is a direct reflection of the drug transport function of the Pgp. In the present study, this system has been utilized to investigate the possibility that antiestrogens and steroid hormones are transported by the Pgp. Antiestrogens such as tamoxifen, metabolites of tamoxifen (4-hydroxytamoxifen and N-desmethyltamoxifen), droloxifen, and toremifene stimulated the Pgp ATPase activity, and the maximum stimulation obtained with these agents equalled the maximal stimulation obtained by the best known MDR chemosensitizer, verapamil. Clomifene, nafoxidine and diethylstilbestrol also stimulated the Pgp ATPase activity, with maximal activations 75, 60 and 45% of the verapamil stimulation, respectively. Different degrees of stimulation of the Pgp ATPase activity were also obtained in the presence of steroid hormones such as progesterone, beta-estradiol, hydrocortisone, and corticosterone. Among these, progesterone is a potent inducer of the Pgp ATPase activity; at 50 microM, this hormone stimulated the Pgp ATPase activity as effectively as verapamil. These results suggest that the antiestrogens and steroid hormones that are known to reverse the multidrug-resistant phenotype do so by directly interacting with Pgp, thus interfering with its anticancer drug-extruding activity.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Antagonistas de Estrogênios/farmacologia , Glicoproteínas de Membrana/metabolismo , Esteroides/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Animais , Proteínas de Transporte/genética , Células Cultivadas , Estradiol/farmacologia , Humanos , Hidrocortisona/farmacologia , Insetos , Glicoproteínas de Membrana/genética , Progesterona/farmacologia , Transfecção , Verapamil/farmacologiaAssuntos
Transporte Biológico , Neurospora/metabolismo , Aminoácidos/metabolismo , Transporte Biológico Ativo , Glucose/metabolismo , Troca Iônica , Metabolismo dos Lipídeos , Neurospora crassa/citologia , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/crescimento & desenvolvimento , Oligomicinas/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Potássio/metabolismo , Ligação Proteica , Esferoplastos/metabolismo , Esporos Fúngicos/citologia , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Sulfatos/metabolismoAssuntos
Neurospora crassa/enzimologia , Neurospora/enzimologia , ATPases Translocadoras de Prótons/isolamento & purificação , Membrana Celular/enzimologia , Parede Celular/ultraestrutura , Centrifugação/métodos , Peso Molecular , Mutação , Neurospora crassa/genética , Neurospora crassa/ultraestrutura , ATPases Translocadoras de Prótons/metabolismoAssuntos
Antiporters , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions , Neurospora crassa/metabolismo , Neurospora/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Transporte Biológico Ativo , Cálcio/metabolismo , Fracionamento Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Métodos , Neurospora crassa/ultraestrutura , PrótonsAssuntos
Adenosina Trifosfatases/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Transporte Biológico Ativo , ATPase Trocadora de Hidrogênio-Potássio , Cinética , Modelos Biológicos , Neurospora crassa/enzimologiaRESUMO
A general hypothesis for the molecular mechanism of membrane transport based on current knowledge of protein structure and the nature of ligand-induced protein conformational changes has recently been proposed [Scarborough, G. A. (1985) Microbiol. Rev. 49, 214-231]. According to this hypothesis, the essential reaction undergone by all proteinaceous transport catalysts is a ligand-induced hinge-bending-type conformational change that results in the transposition of binding-site residues from access on one side of the membrane to access on the other side. Subsequent release and/or alteration of the ligand or ligands that induce the conformational change facilitates the converse conformational change, which returns the binding-site residues to their original position. With this simple cyclic ligand-dependent gating process as a central feature, biochemically orthodox mechanisms for virtually all known transporters are readily conceived. In this article, a chemically explicit model for the molecular mechanism of the F1F0 H+-ATPase/ATP synthases of mitochondria, bacteria, and chloroplasts, formulated within the guidelines of this general transport paradigm, is presented. At least three points of potential interest arise from this exercise. First, with the aid of the model, it is possible to visualize how energy transduction catalyzed by these enzymes might proceed, with no major events left unspecified. Second, explicit possibilities as to the molecular nature of electric field effects on the transport process are raised. And finally, it is shown that enzyme conformational changes, energy-dependent binding-affinity changes, and several other related phenomena as well, need not be taken as evidence of "action at a distance" or indirect energy coupling mechanisms, as is sometimes assumed, because such events are also integral features of the mechanism presented, even though all of the key reactions proposed for both ATP-driven proton translocation and proton translocation-driven ATP synthesis occur at the enzyme active site.
Assuntos
ATPases Translocadoras de Prótons/metabolismo , Animais , Bactérias/enzimologia , Transporte Biológico Ativo , Cloroplastos/enzimologia , Mitocôndrias/enzimologia , Modelos QuímicosRESUMO
The structure of the Neurospora crassa plasma membrane H(+)-ATPase has been investigated using a variety of chemical and physiochemical techniques. The transmembrane topography of the H(+)-ATPase has been elucidated by a direct, protein chemical approach. Reconstituted proteoliposomes containing purified H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin, and the numerous peptides released were purified by HPLC and subjected to amino acid sequence analysis. In this way, seventeen released peptides were unequivocally identified as located on the cytoplasmic side of the membrane, and numerous intervening segments could be inferred to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return between sequences established to be cytoplasmically located. Additionally, three large membrane-embedded segments of the H(+)-ATPase were isolated using our recently developed methods for purifying hydrophobic peptides, and identified by amino acid sequence analysis. This information established the topographical location of virtually all of the 919 residues in the H(+)-ATPase molecule, allowing the formulation of a reasonably detailed model for the transmembrane topography of the H(+)-ATPase polypeptide chain. Separate studies of the cysteine chemistry of the H(+)-ATPase have demonstrated the existence of a single disulfide bridge in the molecule, linking the NH2- and COOH-terminal membrane-embedded domains. And, analyses of the circular dichroism and infrared spectra of the purified H(+)-ATPase have elucidated the secondary structure composition of the molecule. A first-generation model for the tertiary structure of the H(+)-ATPase based on this information and other considerations is presented.
Assuntos
Proteínas Fúngicas/ultraestrutura , ATPases Translocadoras de Prótons/ultraestrutura , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Cisteína/química , Neurospora crassa , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/químicaRESUMO
Large single three-dimensional crystals of the dodecylmaltoside complex of the Neurospora crassa plasma membrane H(+)-ATPase (H(+) P-ATPase) can be grown in polyethylene-glycol-containing solutions optimized for moderate supersaturation of both the protein surfaces and detergent micellar region. Large two-dimensional H(+) P-ATPase crystals also grow on the surface of such mixtures and on carbon films located at such surfaces. Electron crystallographic analysis of the two-dimensional crystals grown on carbon films has recently elucidated the structure of the H(+) P-ATPase at a resolution of 0.8 nm in the membrane plane. The two-dimensional crystals comprise two offset layers of ring-shaped ATPase hexamers with their exocytoplasmic surfaces face to face. Side-to-side interactions between the cytoplasmic regions of the hexamers in each layer can be seen, and an interaction between identical exocytoplasmic loops in opposing hexamer layers holds the two layers together. Detergent rings around the membrane-embedded region of the hexamers are clearly visible, and detergent-detergent interactions between the rings are also apparent. The crystal packing forces thus comprise both protein-protein and detergent-detergent interactions, supporting the validity of the original crystallization strategy. Ten transmembrane helices in each ATPase monomer are well-defined in the structure map. They are all relatively straight, closely packed, moderately tilted at various angles with respect to a plane normal to the membrane surface and average approximately 3.5 nm in length. The transmembrane helix region is connected in at least three places to the larger cytoplasmic region, which comprises several discrete domains separated by relatively wide, deep clefts. Previous work has shown that the H(+) P-ATPase undergoes substantial conformational changes during its catalytic cycle that are not changes in secondary structure. Importantly, the results of hydrogen/deuterium exchange experiments indicate that these conformational changes are probably rigid-body interdomain movements that lead to cleft closure. When interpreted within the framework of established principles of enzyme catalysis, this information on the structure and dynamics of the H(+) P-ATPase molecule provides the basis of a rational model for the sequence of events that occurs as the ATPase proceeds through its transport cycle. The forces that drive the sequence can also be clearly stipulated. However, an understanding of the molecular mechanism of ion transport catalyzed by the H(+) P-ATPase awaits an atomic resolution structure.
Assuntos
Membrana Celular/enzimologia , Cristalização , Neurospora crassa/enzimologia , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/metabolismo , Estrutura Molecular , Conformação Proteica , ATPases Translocadoras de Prótons/metabolismoRESUMO
Living cells require membranes and membrane transporters for the maintenance of life. After decades of biochemical scrutiny, the structures and molecular mechanisms by which membrane transporters catalyze transmembrane solute movements are beginning to be understood. The plasma membrane proton-translocating adenosine triphosphatase (ATPase) is an archetype of the P-type ATPase family of membrane transporters, which are important in a wide variety of cellular processes. The H+-ATPase has been crystallized and its structure determined to a resolution of 8 angstrom in the membrane plane. When considered together with the large body of biochemical information that has been accumulated for this transporter, and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by this enzyme.
Assuntos
Membrana Celular/enzimologia , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Membrana Celular/metabolismo , Cristalização , Cristalografia por Raios X , Transporte de Íons , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Neurospora crassa/enzimologia , Conformação Proteica , ATPases Translocadoras de Prótons/ultraestrutura , Prótons , Relação Estrutura-AtividadeRESUMO
Large single crystals of the dodecylmaltoside (DDM) complex of a polytopic integral membrane transport protein, the Neurospora plasma membrane H(+)-ATPase, have been obtained using an approach that attempts to take into account the possibly radically different physicochemical properties of the protein surfaces and the detergent micellar collar. The overall goal of the crystallization strategy employed was to identify conditions in which the protein surfaces of the DDM-ATPase complex are moderately insoluble and in which the DDM micellar collar is also near its solubility limit. The first step was to screen a variety of commonly used protein precipitants for those that were able to induce the aggregation of pure DDM micelles. The concentration at which any precipitant induced DDM micellar aggregation was hoped to be close to the concentration at which it might induce insolubility of the detergent micellar collar of the DDM-ATPase complex. Of the nine precipitants tried, seven, all polyethylene glycols (PEGs), were able to induce DDM micelle insolubility. The seven PEGs were then tested for their effect on the solubility of the DDM-ATPase complex at a concentration slightly below that necessary to induce DDM micellar aggregation. Three of the PEGs caused extensive precipitation of the ATPase at this concentration and were, therefore, shelved. The other four PEGs did not induce precipitation at the concentration employed and were subsequently used at this concentration for crystallization trials in which the protein concentration was varied. Encouragingly, crystalline plates of the ATPase were obtained for each of the four PEGs tried, indicating that the overall approach may be valid. Unfortunately, the crystals obtained were visibly flawed, suggesting that the correct balance of protein surface and DDM micelle insolubility had not yet been reached. The ionic strength of the crystallization trials was then raised, which was known from other experiments to render the protein surfaces of the ATPase less soluble while having no effect on the DDM micellar aggregation point. For one of the PEGs, PEG 4000, this brought on a new, well formed hexagonal crystal habit. Subsequent optimization of the initial conditions has yielded large single hexagonal crystals of the H(+)-ATPase roughly 0.4 x 0.4 x 0.15 mm in size, holding promise for exploration of the structure of the ATPase by X-ray diffraction analysis.