Influences of energization and nucleotide binding on the reaction of Lucifer Yellow vinyl sulfone with the alpha subunits of the chloroplast ATP synthase

The catalytic portion of the chloroplast ATP synthase (CF(1)) consists of five different polypeptides in the stoichiometry alpha(3)beta(3)gammadeltaepsilon and is structurally asymmetric. Asymmetry is readily apparent in the properties of the six nucleotide binding sites and the single-copy, smaller subunits. Asymmetry is also detected in the alpha subunits by the rapid and covalent binding of Lucifer Yellow vinyl sulfone (LY) to one of the three chemically identical alpha subunits. The binding of LY to a single alpha subunit has allowed the investigation of whether asymmetry in the alpha subunits is a permanent feature of CF(1). The development of an electrochemical proton gradient across illuminated thylakoid membranes and the preincubation of CF(1) in solution with Mg(2+)-ATP were found to alter the LY distribution such that multiple alpha subunits were labeled with LY. Illumination of thylakoid membranes doubled the extent of LY labeling, and fluorescence resonance energy transfer measurements indicated that LY was bound to more than one alpha subunit. Since the change in LY distribution was inhibited by proton ionophores (uncouplers), alteration of alpha conformation by illumination is a result of the generation of a proton gradient. Preincubation of CF(1) in solution with Mg(2+)-ATP had no effect on the extent of LY labeling but resulted in multiple alpha subunits binding LY as determined by fluorescence resonance energy transfer measurements. Adenine nucleotides at substrate level concentrations inhibit the reaction of LY with the alpha subunits. No increase in LY labeling was observed when thylakoids were illuminated under conditions in which CF(1) was catalytically active.     

Labeling of the glycoprotein subunit of (Na,K)ATPase with fluorescent probes

Sodium plus potassium activated adenosinetriphosphatase [(Na,K)ATPase] is composed of a catalytic subunit (alpha) and a glycoprotein subunit (beta) of unknown function. A method has been developed to label the beta subunit of purified dog kidney (Na,K)ATPase with fluorescent probes. The method consists of oxidation of beta-subunit oligosaccharides, reaction of the resulting aldehydes with fluorescent hydrazides, and reduction of the hydrazones and unreacted aldehydes with NaBH4. Two oxidation methods were compared. Simultaneous treatment with neuraminidase and galactose oxidase did not inhibit significantly (Na,K)ATPase activity and allowed insertion of up to 11 mol of probe per mol of beta. In contrast, oxidation of (Na,K)ATPase oligosaccharides with periodate resulted in 50-80% inhibition of the (Na,K)ATPase activity with low or undetectable labeling. Eleven commercial probes and two novel hydrazides were tested for labeling of (Na,K)ATPase treated with galactose oxidase and neuraminidase. Eight probes did not label (Na,-K)ATPase but labeled red cell ghosts oxidized with periodate. Four probes labeled beta specifically but either adsorbed to the membrane tightly, or cross-linked the beta subunits, or formed unstable adducts. Lucifer yellow CH labeled beta specifically without membrane adsorption. Labeling stoichiometries from 1 to 11 mol of lucifer yellow CH per mol of beta were obtained without inhibition of (Na,K)ATPase activity and without significant alteration of the anthroylouabain binding capacity or its association and dissociation kinetics. Anthroylouabain specifically bound to the lucifer-labeled (Na,K)ATPase had a decreased quantum yield, probably due to resonance energy transfer. This suggests that the sites of lucifer attachment on beta are within energy transfer distance from the cardiac glycoside site on alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence-based assay probing regulator of G protein signaling partner proteins

The regulator of G protein signaling (RGS) proteins are one of the essential modulators for the G protein system. Besides regulating G protein signaling by accelerating the GTPase activity of Gα subunits, RGS proteins are implicated in exerting other functions; they are also known to be involved in several diseases. Moreover, the existence of a single RGS protein in plants and its seven-transmembrane domain found in 2003 triggered efforts to unveil detailed structural and functional information of RGS proteins. We present a method for real-time examination of the protein-protein interactions between RGS and Gα subunits. AtRGS1 from plants and RGS4 from mammals were site-directedly labeled with the fluorescent probe Lucifer yellow on engineered cysteine residues and used to interact with different Gα subunits. The physical interactions can be revealed by monitoring the real-time fluorescence changes (8.6% fluorescence increase in mammals and 27.6% in plants); their correlations to functional exertion were shown with a GTPase accelerating activity assay and further confirmed by measurement of K(d). We validate the effectiveness of this method and suggest its application to the exploration of more RGS signaling partner proteins in physiological and pathological studies.     

Conformational changes in the amino-terminal helix of the G protein alpha(i1) following dissociation from Gbetagamma subunit and activation

G protein alpha subunits mediate activation of signaling pathways through G protein-coupled receptors (GPCR) by virtue of GTP-dependent conformational rearrangements. It is known that regions of disorder in crystal structures can be indicative of conformational flexibility within a molecule, and there are several such regions in G protein alpha subunits. The amino-terminal 29 residues of Galpha are alpha-helical only in the heterotrimer, where they contact the side of Gbeta, but little is known about the conformation of this region in the active GTP bound state. To address the role of the Galpha amino-terminus in G-protein activation and to investigate whether this region undergoes activation-dependent conformational changes, a site-directed cysteine mutagenesis study was carried out. Engineered Galpha(i1) proteins were created by first removing six native reactive cysteines to yield a mutant Galpha(i1)-C3S-C66A-C214S-C305S-C325A-C351I that no longer reacts with cysteine-directed labels. Several cysteine substitutions along the amino-terminal region were then introduced. All mutant proteins were shown to be folded properly and functional. An environmentally sensitive probe, Lucifer yellow, linked to these sites showed a fluorescence change upon interaction with Gbetagamma and with activation by AlF(4)(-). Other fluorescent probes of varying charge, size, and hydrophobicity linked to amino-terminal residues also revealed changes upon activation with bulkier probes reporting larger changes. Site-directed spin-labeling studies showed that the N-terminus of the Galpha subunit is dynamically disordered in the GDP bound state, but adopts a structure consistent with an alpha-helix upon interaction with Gbetagamma. Interaction of the resulting spin-labeled Galphabetagamma with photoactivated rhodopsin, followed by rhodopsin-catalyzed GTPgammaS binding, caused the amino-terminal domain of Galpha to revert to a dynamically disordered state similar to that of the GDP-bound form. Together these results suggest conformational changes occur in the amino-termini of Galpha(i) proteins upon subunit dissociation and upon activating conformational changes. These solution studies reveal insights into conformational changes that occur dynamically in solution.     

Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes

Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature- dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine- labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.     

Fluorescence energy transfer between ligand binding sites on chloroplast coupling factor 1.

The method of fluorescence energy transfer is used to measure the distance from the tight nucleotide binding sites to the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reactive sites on solubilized spinach chloroplast coupling factor 1 (CF1). The fluorescent adenine nucleotide analogs 1,N-6-ethenoadenosine diphosphate and 1,N-6-ethenoadenylyl imidodiphosphate were used as donors and 4-nitrobenzo-2-oxa-1,3-diazole bound to a tyrosine group and to an amino group were used as acceptors of energy transfer. Using three different donor-acceptor pairs, the distance measured varied from 38 to 43 A assuming both donor sites are equidistant from the acceptor site. A model is proposed for the location of the tight nucleotide binding sites and the active site on the alpha and beta subunits of CF1.     

Role of the ATP synthase alpha-subunit in conferring sensitivity to tentoxin.

Tentoxin, produced by phytopathogenic fungi, selectively affects the function of the ATP synthase enzymes of certain sensitive plant species. Binding of tentoxin to a high affinity (K(i) approximately 10 nM) site on the chloroplast F(1) (CF(1)) strongly inhibits catalytic function, whereas binding to a second, lower affinity site (K(d) > 10 microM) leads to restoration and even stimulation of catalytic activity. Sensitivity to tentoxin has been shown to be due, in part, to the nature of the amino acid residue at position 83 on the catalytic beta subunit of CF(1). An aspartate in this position is required, but is not sufficient, for tentoxin inhibition. By comparison with the solved structure of mitochondrial F(1) [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], Asp83 is probably located at an interface between alpha and beta subunits on CF(1) where residues on the alpha subunit could also participate in tentoxin binding. A hybrid core F(1) enzyme assembled with beta and gamma subunits of the tentoxin-sensitive spinach CF(1), and an alpha subunit of the tentoxin-insensitive photosynthetic bacterium Rhodospirillum rubrum F(1) (RrF(1)), was stimulated but not inhibited by tentoxin [Tucker, W. C., Du, Z., Gromet-Elhanan, Z. and Richter, M. L. (2001) Eur. J. Biochem. 268, 2179-2186]. In this study, chimeric alpha subunits were prepared by introducing short segments of the spinach CF(1) alpha subunit from a poorly conserved region which is immediately adjacent to beta-Asp83 in the crystal structure, into equivalent positions in the RrF(1) alpha subunit using oligonucleotide-directed mutagenesis. Hybrid enzymes containing these chimeric alpha subunits had both the high affinity inhibitory tentoxin binding site and the lower affinity stimulatory site. Changing beta-Asp83 to leucine resulted in loss of both inhibition and stimulation by tentoxin in the chimeras. The results indicate that tentoxin inhibition requires additional alpha residues that are not present on the RrF(1) alpha subunit. A structural model of a putative inhibitory tentoxin binding pocket is presented.     

Molecular characterization of two point mutants in the chloroplast atpB gene of the green alga Chlamydomonas reinhardtii defective in assembly of the ATP synthase complex

Two point mutants of Chlamydomonas reinhardtii, previously found by recombination and complementation analysis to map in the chloroplast atpB gene encoding the beta subunit of the CF1/CF0 ATP synthase, are here shown to be missense alterations near the 5' end of that gene. One mutant (ac-u-c-2-9) has a change at amino acid position 47 of the beta subunit from leucine (CTA) to arginine (CGA). In the second mutant (ac-u-c-2-29), the codon AAA (lysine) is changed to AAC (asparagine) at position 154. Spontaneous revertants of each mutant were isolated that restore the original wild type base pair. Northern analysis of total RNA and in vivo pulse labeling followed by immunoprecipitation reveals that both mutant atpB genes are transcribed and translated normally. However, immunoblots show that the amount of beta subunit associated with mutant thylakoids is only approximately 3% of that seen in wild type and that the CF1 alpha and gamma subunits are missing entirely. The disruption of ATP synthase complex assembly in these mutants is much more severe than in Escherichia coli beta subunit gene point mutants, which retain significant amounts of alpha and beta subunits on their membranes (Noumi, T., Oka, N., Kanazawa, H., and Futai, M. (1986) J. Biol. Chem. 261, 7070-7075). These results support the hypothesis that there are differences in assembly of the ATP synthase between E. coli and chloroplasts. In particular they indicate that beta must be present for assembly of the alpha and gamma subunits of CF1 onto chloroplast membranes.     

Organization of calcium channel beta1a subunits in triad junctions in skeletal muscle

In skeletal muscle, dihydropyridine receptors (DHPRs) in the plasma membrane interact with the type 1 ryanodine receptor (RyR1) at junctions with the sarcoplasmic reticulum. This interaction organizes junctional DHPRs into groups of four termed tetrads. In addition to the principle alpha1S subunit, the beta1a subunit of the DHPR is also important for the interaction with RyR1. To probe this interaction, we measured fluorescence resonance energy transfer (FRET) of beta1a subunits labeled with cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP). Expressed in dysgenic (alpha1S-null) myotubes, YFP-beta1a-CFP and CFP-beta1a-YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, beta1a does not appear to bind to other cellular proteins in the absence of alpha1S. FRET efficiencies for these cytoplasmic beta1a subunits were approximately 6-7%, consistent with the idea that <10 nm separates the N and C termini. After coexpression with unlabeled alpha1S (in dysgenic or beta1-null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In beta1-null myotubes, FRET efficiencies of doubly labeled beta1a in puncta were similar to those of the same constructs diffusely distributed in the cytoplasm and appeared to arise intramolecularly, since no FRET was measured when mixtures of singly labeled beta1a (CFP or YFP at the N or C terminus) were expressed in beta1-null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent beta1a subunits are located >10 nm from one another.     

Gap junction permeability: selectivity for anionic and cationic probes

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 ?, charge -2), carboxyfluorescein (CF; 8.2 ?; -2), and Alexa Fluor350 (AF350, 5.4 ?; -1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 ?, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 ?, +1). Comparison of probe permeability relative to K(+) revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.     

Effect of proteolytic digestion on the Ca2+-ATPase activity and subunits of latent and thiol-activated chloroplast coupling factor 1

The activation by proteases of the Ca2+-dependent ATPase of chloroplast coupling factor 1 (CF1) has been investigated. Using low concentrations of papain and trypsin, the increase in ATPase activity and the degradation of the five subunits of CF1 were compared. Sodium dodecyl sulfate-gel electrophoresis of protease-treated CF1 revealed that the delta subunit was very rapidly degraded and that the alpha and beta subunits were clipped. The gamma and epsilon subunits were more resistant to digestion. The modification of the alpha subunit of latent CF1 most closely correlated with the activation of Ca2+-ATPase activity. Trypsin treatment of dithiothreitol-activated CF1 resulted in a very rapid increase in Ca2+-ATPase activity and a corresponding rapid cleavage of the gamma subunit to a 25,000-dalton species. With more prolonged treatment, the 25,000-dalton species was cleaved to fragments of 14,000 and 11,000-daltons. Dithiothreitol treatment did not alter the rate of attack on the other subunits. The gamma subunit of heat-activated CF1 was also more susceptible to protease digestion. The increased protease sensitivity of the gamma subunit of soluble CF1 after treatment with dithiothreitol or heat mimics the increased protease sensitivity of the gamma subunit of bound CF1 when thylakoids are treated with trypsin during illumination (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5915-5920). These results suggest that the conformational changes that occur when purified CF1 is exposed to dithiothreitol are similar to those that CF1 bound to thylakoid membranes undergoes under illumination.     

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.     

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.     

Alteration of the nucleotide-binding site asymmetry of chloroplast coupling factor 1 by catalysis

Fluorescence resonance energy transfer was used to show that ATP hydrolysis induces a change in the properties of two nucleotide-binding sites in isolated chloroplast coupling factor 1 (CF1). The fluorescence donor was Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide- 3,6-disulfonate), covalently bound to a unique site on the alpha subunit between nucleotide-binding sites 2 and 3. The fluorescence acceptor was the ATP analog 2'(3')-trinitrophenyladenosine 5'-triphosphate (TNP-ATP), incorporated specifically into nucleotide-binding site 1. Energy transfer from Lucifer Yellow to TNP-ATP in site 1 was greater if catalysis occurred before TNP-ATP was incorporated than if no catalysis occurred, indicating that one of the nucleotide-binding sites near the Lucifer Yellow had changed its properties to those of site 1 as a result of catalysis. The amount of energy transfer increased with the degree of substrate excess during catalysis, as expected if catalysis were required for the new site 1 location. ADP, which binds to CF1, but is not a substrate for hydrolysis, caused little energy transfer. Titration of site 3 with TNP-ATP showed greater energy transfer from Lucifer Yellow when catalysis had not occurred, indicating that sites 1 and 3 switched properties as a result of catalysis. The amount of energy transfer declined exponentially with time between removal of substrate and addition of TNP-ATP to site 1, with a half-time of 1.5-2 h at room temperature. This result suggests that the change that results in switching of nucleotide-binding sites 1 and 3 relaxes in the absence of substrate. Our results show that the asymmetry of the nucleotide-binding sites of CF1 is not a permanent feature of the molecule.     

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively.     

In vitro synthesis and assembly of the peripheral subunits of coupling factor CF1 (alpha and beta) by thylakoid-bound ribosomes

Bispecific antisera were prepared to a mixture of thylakoid membrane polypeptides 4.1 and 4.2. The identity of these polypeptides as the alpha and beta subunits of coupling factor (CF1) was established based on the cross-reactivity of the antisera toward CF1 from peas and by an analysis of the thm-24 mutant of Chlamydomonas which lacks the CF1 ATPase. Photochemical labeling of thylakoid membranes with hydrophobic and hydrophilic fluorescent probes indicated that these polypeptides did not significantly penetrate the membrane bilayer. Immunoprecipitation of the translation products of thylakoid-bound and soluble ribosomes showed the thylakoids to be the major site of synthesis of the polypeptides. Immunoprecipitation of the products of translation of total cellular RNA in a reticulocyte lysate showed no evidence for substantially higher molecular weight precursors. Further analysis of the thylakoid-bound synthesis of alpha and beta revealed that some of the in vitro synthesized polypeptides had been incorporated into the CF0-CF1 complex based on their release from membranes with trypsin and copurification with the CF0-CF1 ATPase.     

The domain structure of tryptophan synthase. A neutron scattering study

Tryptophan synthase from Escherichia coli is a complex of two alpha subunits and two beta subunits. Small-angle neutron scattering involving deuterium-labelled isomers revealed the quaternary structure of the enzyme at the level of the beta 2 subunit and the two structural domains P1 and P2 which constitute the alpha subunits. Within the alpha 2 beta 2 complex, the two alpha subunits are completely separated. They are situated on opposite sides of the beta 2 subunit. The most probable distance between the two alpha protomers is 10.5 +/- 1 nm; the nearest distance is 5.8 +/- 0.5 nm, and the largest distance is 13.5 +/- 0.5 nm. The two domains of the same alpha subunit are intimately juxtaposed. The distances between two like or unlike domains belonging to opposite alpha subunits are roughly equal. All domains exhibit about equal distances to the beta 2 subunit which is situated in the centre of the complex. Thus the cleft between P1 and P2, which probably contains the active site of the alpha subunit, makes intimate contact with the beta 2 subunit. Neutron scattering allows us to determine the shape of the beta 2 subunit within the complex. Comparison with the free dimer suggests a conformational change, upon assembly, from an elongated into a more compact form.     

Delineation of bacterial luciferase aldehyde site by bifunctional labeling reagents

Previously we have established that a highly reactive cysteinyl group on the alpha subunit is at the aldehyde site of the (alpha beta) dimeric Vibrio harveyi luciferase. Three isomeric bifunctional reagents have been synthesized and used to further delineate the luciferase aldehyde site. These probes differ in their relative positions of and distances between the two functional groups active in chemical and photochemical labelings, respectively. Each of the probes can effectively and reversibly inactivate luciferase by forming a disulfide linkage primarily to the reactive cysteinyl residue. Upon subsequent photolysis, a diazoacetate arm in each probe was activated for photochemical labeling of amino acid residues within reach. After reductive regeneration of the reactive cysteinyl residue, 0.35-0.40 probe per dimeric luciferase was found to have been photochemically incorporated, correlating well with the degree of irreversible enzyme inactivation. Low but significant amounts of the three isomeric probes initially attached to the alpha reactive cysteine through a disulfide have been found to photochemically tag certain residues on beta. The latter residues are estimated to be no more than 8-11 A away from the alpha reactive cysteine. Thus the reactive cysteinyl residue, and hence the aldehyde site, must be at or near the alpha beta subunit interface. Furthermore, the structural integrity of the microenvironment surrounding this reactive cysteinyl residue is crucial to luciferase activity. An HPLC method for the isolation of luciferase alpha and beta subunits has also been developed.     

Intracellular Domains of Mouse Connexin26 and -30 Affect Diffusional and Electrical Properties of Gap Junction Channels

To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins. These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30 cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance and voltage gating of the channels. Received: 26 July 2000/Revised: 15 February 2001