Photochemical construction of coumarin fluorophore on affinity-anchored protein

A simple and unique construction of a coumarin fluorophore on a prey protein has been achieved by sequential photoreactions using a nonfluorescent bait protein. The bait protein was first modified with a novel diazirine-based photo-cross-linker containing an o-hydroxycinnamoyl unit. The strategy involves two wavelength-dependent photoreactions: photolysis of the diazirine group and E to Z photoisomerization of the cinnamoyl group. The cross-linked interacting partners were cleaved by the consecutive steps of photoisomerization and thermal lactonization accompanied with the creation of a coumarin derivative on the prey protein as a fluorescent tag. Finally, the methodology was successfully applied for coumarin labeling of antilysozymes expressed on living B cells by using photoactivatable lysozymes.     

Rapidly photoactivatable ATP probes for specific labeling of tropomyosin within the actomyosin protein complex

Tropomyosin-specific photoaffinity adenosine triphosphate (ATP) probes have been first developed, in which a diazirine moiety is incorporated into the γ-phosphate group as a rapidly carbene-generating photophore. These probes clearly labeled tropomyosin in the presence of other actomyosin components, that is, myosin, actin, and troponins. The specific labeling of tropomyosin was easily identified by selective trapping of the photo-incorporated ATP probe on Fe³⁺-immobilized metal ion affinity chromatography (IMAC) beads. The characteristic nature of tropomyosin-specific photocross-linking was further confirmed with a biotin-carrying derivative of the ATP probe. These data suggest that the tropomyosin on the actin filament assembly is located in close proximity to the ATP binding cavity of myosin.     

Protein phosphorylation regulates in vitro spinach chloroplast petD mRNA 3′-untranslated region stability,processing, and degradation

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3′-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3′-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3′-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3′-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3′-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3′-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3′-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3′-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3′-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast.     

ATP is required for the binding of precursor proteins to chloroplasts

One of the first steps in the transport of nuclear-encoded, cytoplasmically synthesized precursor proteins into chloroplasts is a specific binding interaction between precursor proteins and the surface of the organelle. Although protein translocation into chloroplasts requires ATP hydrolysis, binding is generally thought to be energy independent. A more detailed investigation of precursor binding to the surface of chloroplasts showed that ATP was required for efficient binding. Protein translocation is known to require relatively high levels (1 mM or more) of ATP. As little as 50-100 microM ATP caused significant stimulation of precursor binding over controls with no ATP. Several different precursors were tested and all showed increased binding upon addition of low levels of ATP. Nonhydrolyzable analogs of ATP did not substitute for ATP, indicating that ATP hydrolysis was required for binding. A protonmotive force was not involved in the energy requirement for binding. Other (hydrolyzable) nucleotides could substitute for ATP but were less effective at stimulating binding. Binding was stimulated by ATP generated inside chloroplasts even when an ATP trap was present to destroy external ATP. We conclude that internal ATP is required for stimulation of precursor binding to chloroplasts.     

Bacterial Sec Protein Transport Is Rate-limited by Precursor Length: A Single Turnover Study

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.     

Onognaphalin,a further 5-methyl coumarin from Onoseris gnaphalioides

From the roots of Onoseris gnaphalioides in addition to a known 5-methyl coumarin a new type was isolated. The aerial parts contain a glucoside of 4-hydroxy-5-methyl coumarin. From Gerbera ambigua a compound related to the possible precursor of the 5-methyl coumarins was isolated.     

The 24 kDa N-terminal sub-domain of the DNA gyrase B protein binds coumarin drugs

A number of lines of evidence suggest that the N-terminal sub-domain of the DNA gyrase B protein contains the binding site for the coumarin antibiotics. We have engineered a clone which encodes a 24 kDa protein which represents this domain. Bacteria which overproduce this protein show an elevated level of resistance to coumarins, suggestive of binding of the 24 kDa protein to the drugs In vivo. In vitro we find that the 24 kDa protein does not interact with the gyrase A or B proteins or with DNA, and fails to hydrolyse ATP or show significant binding to ATP, ADP or ADPNP. However, we show that the 24 kDa protein binds coumarin drugs as tightly as the Intact B protein. A number of experiments suggest that the Interaction of the coumarins with the protein is predominantly hydrophobic in nature.     

Development of carbamate-tethered coumarins as phototriggers for caged nicotinamide

The syntheses of 7-diethylaminocoumarin- or modified DEACM-nicotinamide and 6-bromo-7-methoxycoumarin- or BMCM-nicotinamide have been accomplished by reaction of nicotinoyl isocyanate with the corresponding coumarin allylic alcohol derivatives. The resulting compounds contain an N-acyl O-alkyl carbamate as a new type of linkage for the caging of nicotinamide with a coumarin phototrigger, which undergoes cleavage upon photolysis. Our design of specific caged-nicotinamides was based upon NBO and TD-FT calculations to predict absorption wavelengths and photocleavage potential. This work provides a potentially general method for the caging of amides with coumarin photolabile protecting groups.     

The carboxyl-terminal two-thirds of the ADP/ATP carrier polypeptide contains sufficient information to direct translocation into mitochondria

The precursor of the mitochondrial inner membrane protein ADP/ATP carrier is cytoplasmically synthesized without an amino-terminal peptide extension. We constructed a truncated precursor lacking the 103 amino acids from the amino terminus (about a third of the protein). Import of the truncated precursor into mitochondria showed the import characteristics of the authentic precursor, including nucleoside triphosphate dependence, requirement for a protease-sensitive component on the mitochondrial surface, two-step specific binding to the outer membrane, and membrane potential-dependent translocation into the inner membrane. We conclude that, in contrast to all other mitochondrial precursor proteins studied so far, domains of the ADP/ATP carrier distant from the amino terminus can carry specific targeting information for transport into mitochondria.     

Chromogenic diazirine: A new spectrophotometric approach for photoaffinity labeling

[[2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]]phenoxy]acetic acid and its derivatives have been synthesized as a special carbene precursor with a chromogenic group. Photolysis of the diazirine in methanol and cyclohexane gave intermolecular O---H and C---H insertion products, respectively. Spectroscopic properties of the diazirine derivatives and the photo-products revealed that irradiation and detection can be performed in a spectral region where the absorption due to most biological macromolecules is negligible. The application of this reagent will provide a useful approach for simple spectrophotometric detection of labeled products without recourse to conventional radioactive techniques in the photoaffinity labeling methodology.     

Membrane topography of the coupling ion binding site in Na+-translocating F1F0 ATP synthase.

A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na(+)-translocating F(1)F(0) ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na(+) ions but not in its presence, indicating the specific reaction with the Na(+) binding c-Glu(65) residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu(65) was therefore in close contact with phospholipid but does not contact subunit a. Na(+)in/(22)Na(+)out exchange activity of the ATP synthase was not affected by modifying the c-Glu(65) sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na(+) ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospholipases C and A(2) and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu(65) is located within the core of the membrane.     

Stable association of chloroplastic precursors with protein translocation complexes that contain proteins from both envelope membranes and a stromal Hsp100 molecular chaperone.

Cytoplasmically synthesized precursors interact with translocation components in both the outer and inner envelope membranes during transport into chloroplasts. Using co-immunoprecipitation techniques, with antibodies specific to known translocation components, we identified stable interactions between precursor proteins and their associated membrane translocation components in detergent-solubilized chloroplastic membrane fractions. Antibodies specific to the outer envelope translocation components OEP75 and OEP34, the inner envelope translocation component IEP110 and the stromal Hsp100, ClpC, specifically co-immunoprecipitated precursor proteins under limiting ATP conditions, a stage we have called docking. A portion of these same translocation components was co-immunoprecipitated as a complex, and could also be detected by co-sedimentation through a sucrose density gradient. ClpC was observed only in complexes with those precursors utilizing the general import apparatus, and its interaction with precursor-containing translocation complexes was destabilized by ATP. Finally, ClpC was co-immunoprecipitated with a portion of the translocation components of both outer and inner envelope membranes, even in the absence of added precursors. We discuss possible roles for stromal Hsp100 in protein import and mechanisms of precursor binding in chloroplasts.     

A quantitative method for the measurement of cellular guanosine triphosphate pool specific activities

Studies on quantitation of RNA synthesis in eucaryotic cells have frequently used adenosine as the radioactively labeled precursor, largely because of the convenience of the firefly luciferin-luciferase assay in measuring ATP pool specific activity (1,2). This could result in some difficulties if the addition of poly(A) to the 3′ OH end of RNA represents a significant portion of total incorporation, as is the case in sea-urchin embryos (3). In addition, in some cases, the ATP pool may be large enough to prevent the use of adenosine as an effective labeling agent. Hence, a simple and sensitive method for the determination of the specific activity of the other nucleic acid precursor pools would be of value.Although the crystalline luciferase is specific for ATP, extracts of firefly lanterns most commonly used for quantitating ATP (4–9) also exhibit activity with other ribonucleoside triphosphates, adenosine tetraphosphate, ADP, and the deoxyribonucleoside triphosphates. This activity is due to the presence of contaminating enzymes such as nucleoside 5′-diphosphate kinase and adenylate kinase which catalyze the formation of ATP from these nucleotides and trace amounts of ADP, also present in the extracts (10–13). Recently, Manandhar and Van Dyke (14) have reported a procedure for quantitating picomole levels of GTP with a crude extract of firefly lanterns. In the present study, we have adapted their procedure to develop an assay for GTP pool specific activity in Xenopus laevis oocytes microinjected with [8-³H]GTP. Our assay may be extended to the analysis of any nucleoside triphosphate pool, provided that an adequate chromatography system is available for the separation of the extracted nucleotides.     

Reinvestigation of the requirement of cytosolic ATP for mitochondrial protein import

Protein import into mitochondria requires the energy of ATP hydrolysis inside and/or outside mitochondria. Although the role of ATP in the mitochondrial matrix in mitochondrial protein import has been extensively studied, the role of ATP outside mitochondria (external ATP) remains only poorly characterized. Here we developed a protocol for depletion of external ATP without significantly reducing the import competence of precursor proteins synthesized in vitro with reticulocyte lysate. We tested the effects of external ATP on the import of various precursor proteins into isolated yeast mitochondria. We found that external ATP is required for maintenance of the import competence of mitochondrial precursor proteins but that, once they bind to mitochondria, the subsequent translocation of presequence-containing proteins, but not the ADP/ATP carrier, proceeds independently of external ATP. Because depletion of cytosolic Hsp70 led to a decrease in the import competence of mitochondrial precursor proteins, external ATP is likely utilized by cytosolic Hsp70. In contrast, the ADP/ATP carrier requires external ATP for efficient import into mitochondria even after binding to mitochondria, a situation that is only partly attributed to cytosolic Hsp70.     

Energetics-based discovery of protein-ligand interactions on a proteomic scale

Biochemical functions of proteins in cells frequently involve interactions with various ligands. Proteomic methods for the identification of proteins that interact with specific ligands such as metabolites, signaling molecules, and drugs are valuable in investigating the regulatory mechanisms of cellular metabolism, annotating proteins with unknown functions, and elucidating pharmacological mechanisms. Here we report an energetics-based target identification method in which target proteins in a cell lysate are identified by exploiting the effect of ligand binding on their stabilities. Urea-induced unfolding of proteins in cell lysates is probed by a short pulse of proteolysis, and the effect of a ligand on the amount of folded protein remaining is monitored on a proteomic scale. As proof of principle, we identified proteins that interact with ATP in the Escherichia coli proteome. Literature and database mining confirmed that a majority of the identified proteins are indeed ATP-binding proteins. Four identified proteins that were previously not known to interact with ATP were cloned and expressed to validate the result. Except for one protein, the effects of ATP on urea-induced unfolding were confirmed. Analyses of the protein sequences and structure models were also employed to predict potential ATP binding sites in the identified proteins. Our results demonstrate that this energetics-based target identification approach is a facile method to identify proteins that interact with specific ligands on a proteomic scale.     

A protein kinase family in Arabidopsis phosphorylates chloroplast precursor proteins

A serine/threonine protein kinase that is able to phosphorylate chloroplast-destined precursor proteins was purified from leaf extract of Arabidopsis thaliana and was identified by mass spectrometry. The protein kinase, encoded by AT2G17700, belongs to a small protein family comprising in addition AT4G35780 and AT4G38470. All three proteins were expressed heterologously in Escherichia coli and characterized with regard to their properties in precursor protein phosphorylation. They were able to phosphorylate several chloroplast-destined precursor proteins within their cleavable presequences. In contrast, a mitochondria-destined precursor protein was not a substrate for these kinases. For all three enzymes, the phosphorylation reaction was specific for ATP with apparent K(m) values between 14 and 67 microM. They did not utilize other NTPs nor were those able to compete for ATP in the reaction. An excess of ADP was able to inhibit ATP-dependent phosphorylation. Furthermore, all three kinases exhibited autophosphorylation. The protein kinases described here could represent subunits of a regulatory network involved in the cytosolic events of chloroplast protein import.     

Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (k_cat/k_m) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid mono-layer, in contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.     

Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix.

ATP is needed for the import of precursor proteins into mitochondria. However, the role of ATP and its site of action have been unclear. We have now investigated the ATP requirements for protein import into the mitochondrial matrix. These experiments employed an in vitro system that allowed ATP levels to be manipulated both inside and outside the mitochondrial inner membrane. Our results indicate that there are two distinct ATP requirements for mitochondrial protein import. ATP in the matrix is always needed for complete import of precursor proteins into this compartment, even when the precursors are presented to mitochondria in an unfolded conformation. In contrast, the requirement for external ATP is precursor-specific; depletion of external ATP strongly inhibits import of some precursors but has little or no effect with other precursors. A requirement for external ATP can often be overcome by denaturing the precursor with urea. We suggest that external ATP promotes the release of precursors from cytosolic chaperones, whereas matrix ATP drives protein translocation across the inner membrane.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

gyrB mutations which confer coumarin resistance also affect DNA supercoiling and ATP hydrolysis by Escherichia coli DNA gyrase

Coumarins are inhibitors of the ATP hydrolysis and DNA supercoiling reactions catalysed by DNA gyrase. Their target is the B subunit of gyrase (GyrB), encoded by the gyrB gene. The exact mode and site of action of the drugs is unknown. We have identified four mutations conferring coumarin resistance to Escherichia coli: Arg-136 to Cys, His or Ser and Gly-164 to Val. In vitro, the ATPase and supercoiling activities of the mutant GyrB proteins are reduced relative to the wild-type enzyme and show resistance to the coumarin antibiotics. Significant differences in the susceptibility of mutant GyrB proteins to inhibition by either chlorobiocin and novobiocin or coumermycin have been found, suggesting wider contacts between coumermycin and GyrB. We discuss the significance of Arg-136 and Gly-164 in relation to the notion that coumarin drugs act as competitive inhibitors of the ATPase reaction.