Determinants of the pH of the Golgi complex

The factors contributing to the establishment of the steady state Golgi pH (pH(G)) were studied in intact and permeabilized mammalian cells by fluorescence ratio imaging. Retrograde transport of the nontoxic B subunit of verotoxin 1 was used to deliver pH-sensitive probes to the Golgi complex. To evaluate whether counter-ion permeability limited the activity of the electrogenic V-ATPase, we determined the concentration of K(+) in the lumen of the Golgi using a null point titration method. The [K(+)] inside the Golgi was found to be close to that of the cytosol, and increasing its permeability had no effect on pH(G). Moreover, the capacity of the endogenous counter-ion permeability exceeded the rate of H(+) pumping, implying that the potential across the Golgi membrane is negligible and has little influence on pH(G). The V-ATPase does not reach thermodynamic equilibrium nor does it seem to be allosterically inactivated at the steady state pH(G). In fact, active H(+) pumping was detectable even below the resting pH(G). A steady state pH was attained when the rate of pumping was matched by the passive backflux of H(+) (equivalents) or "leak." The nature of this leak pathway was investigated in detail. Neither vesicular traffic nor H(+)/cation antiporters or symporters were found to contribute to the net loss of H(+) from the Golgi. Instead, the leak was sensitive to voltage changes and was inhibited by Zn(2+), resembling the H(+) conductive pathway of the plasma membrane. We conclude that a balance between an endogenous leak, which includes a conductive component, and the H(+) pump determines the pH at which the Golgi lumen attains a steady state.     

Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia.

The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.     

Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance

Rat liver Golgi vesicles were isolated by differential and density gradient centrifugation. A fraction enriched in galactosyl transferase and depleted in plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal markers was found to contain an ATP-dependent H+ pump. This proton pump was not inhibited by oligomycin but was sensitive to N-ethyl maleimide, which distinguishes it from the F0-F1 ATPase of mitochondria. GTP did not induce transport, unlike the lysosomal H+ pump. The pump was not dependent on the presence of potassium nor was it inhibited by vanadate, two of the characteristics of the gastric H+ ATPase. Addition of ATP generated a membrane potential that drove chloride uptake into the vesicles, suggesting that Golgi membranes contain a chloride conductance in parallel to an electrogenic proton pump. These results demonstrate that Golgi vesicles can form a pH difference and a membrane potential through the action of an electrogenic proton translocating ATPase.     

H+/ATP stoichiometry of proton pumps from Neurospora crassa and Escherichia coli

A kinetic method has been used to measure the apparent stoichiometry of H+ ions translocated per ATP split by membrane-bound [H+]-ATPases. In this method, membrane vesicles are suspended in well-buffered medium, ATP is added, and a fluorescent probe of delta pH (acridine orange) is used to detect the formation of a steady-state pH gradient. At the steady state, it is assumed that proton pumping in one direction is exactly balanced by the leak of protons in the opposite direction. The pump is then rapidly turned off by the addition of an appropriate inhibitor, and the initial rate of relaxation of delta pH is used to infer the pump rate. This rate is divided by the rate of ATP hydrolysis, measured under the same condition, to give the apparent H+/ATP stoichiometry. The method has been applied to two different [H+]-ATPases, the plasma-membrane ATPase of Neurospora (a Mr = 100,000 integral membrane protein) and the ATPase of Escherichia coli (which belongs to the F0F1 group). The Neurospora ATPase displayed an apparent stoichiometry close to 1 H+/ATP (0.82-1.23), in agreement with previous estimates from electrophysiological measurements on whole cells. In contrast, the E. coli ATPase yielded an apparent stoichiometry close to 2 H+/ATP (1.90), consistent with several published values obtained by both kinetic and thermodynamic methods for bacterial, mitochondrial, and chloroplast ATPases.     

Golgi alkalinization by the papillomavirus E5 oncoprotein

The E5 oncoprotein of bovine papillomavirus type I is a small, hydrophobic polypeptide localized predominantly in the Golgi complex. E5-mediated transformation is often associated with activation of the PDGF receptor (PDGF-R). However, some E5 mutants fail to induce PDGF-R phosphorylation yet retain transforming activity, suggesting an additional mechanism of action. Since E5 also interacts with the 16-kD pore-forming subunit of the vacuolar H(+)-ATPase (V-ATPase), the oncoprotein could conceivably interfere with the pH homeostasis of the Golgi complex. A pH-sensitive, fluorescent bacterial toxin was used to label this organelle and Golgi pH (pH(G)) was measured by ratio imaging. Whereas pH(G) of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH approximately 7.0). The Golgi buffering power and the rate of H(+) leakage were found to be comparable in control and transfected cells. Instead, the E5-induced pH differential was attributed to impairment of V-ATPase activity, even though the amount of ATPase present in the Golgi complex was unaltered. Mutations that abolished binding of E5 to the 16-kD subunit or that targeted the oncoprotein to the endoplasmic reticulum abrogated Golgi alkalinization and cellular transformation. Moreover, transformation-competent E5 mutants that were defective for PDGF-R activation alkalinized the Golgi lumen. Neither transformation by sis nor src, two oncoproteins in the PDGF-R signaling pathway, affected pH(G). We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.     

Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies

The possibility of obtaining from any antibody a fluorescent conjugate which responds to the binding of the antigen by a variation of its fluorescence, would be of great interest in the analytical sciences and for the construction of protein chips. This possibility was explored with antibody mAbD1.3 directed against hen egg white lysozyme. Rules of design were developed to identify the residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. These rules were based on: the target residue belonging to a topological neighbourhood of the antigen in the structure of the complex between antibody and antigen; its absence of functional importance for the interaction with the antigen; and its solvent accessibility in the structure of the free antibody. Seventeen conjugates between the single-chain variable fragment scFv of mAbD1.3 and an environment-sensitive fluorophore were constructed. For six of the ten residues which fully satisfied the design rules, the relative variation of the fluorescence intensity between the free and bound states of the conjugate was comprised between 12 and 75% (in non-optimal buffer), and the affinity of the conjugate for lysozyme remained unchanged relative to the parental scFv. In contrast, such results were true for only one of the seven residues which failed to satisfy one of the rules and were used as controls. One of the conjugates was studied in more detail. Its fluorescence increased proportionally to the concentration of lysozyme in a nanomolar range, up to 90% in a defined buffer, and 40% in serum. This increase was specific for hen egg lysozyme and it was not observed with a closely related protein, turkey egg lysozyme. The residues which gave operational conjugates (six in V(L) and one in V(H)), were located in the immediate vicinity of residues which are functionally important, along the sequence of FvD1.3. The results suggest rules of design for constructing antigen-sensitive fluorescent conjugates from any antibody, in the absence of structural data.     

Breaking up and making up: The secret life of the vacuolar H+‐ATPase

The vacuolar ATPase (V‐ATPase; V₁V_o‐ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin‐coated vesicles. V‐ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V‐ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V‐ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high‐resolution structure determination by cryo electron microscopy and X‐ray crystallography together with sophisticated single‐molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V‐ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.     

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.     

Production and characterization of a monoclonal antibody to vacuolar H+ATPase of renal epithelia

A monoclonal antibody to vacuolar H+ATPase isolated from bovine kidney medulla was produced and characterized by immunoprecipitation and immunocytochemistry. The antibody, immobilized on beads, specifically immunoprecipitated both solubilized N-ethylmaleimide-sensitive ATPase activity and proton-transporting vesicles from renal microsomes; control experiments with an "irrelevant" monoclonal antibody showed no immunoprecipitated activity. By fluorescent immunocytochemistry, the antibody stained the membranes of intracellular vacuolar compartments in LLC-PK1 cells. Immunocytochemical staining showed that the monoclonal antibody colocalized partially with N-(3-[2,4-dinitrophenyl)amino)propyl)-N-(3-amino-propyl)methylamine, a probe for acidic compartments, with the endocytic markers dextran and transferrin, with the lysosomal probe alpha 2-macroglobulin, and with clathrin. The anti-vacuolar H+ATPase antibody showed no colocalization with staining for mitochondrial H+ATPase. The anti-vacuolar H+ATPase antibody should serve as a specific probe for examining the distribution and dynamics of the vacuolar proton pump in renal epithelial cells.     

Mechanisms of pH regulation in the regulated secretory pathway

A precise pH gradient between organelles of the regulated secretory pathway is required for sorting and processing of prohormones. We studied pH regulation in live endocrine cells by targeting biotin-based pH indicators to cellular organelles expressing avidin-chimera proteins. In AtT-20 cells, we found that steady-state pH decreased from the endoplasmic reticulum (ER) (pH(ER) = 7.4 +/- 0.2, mean +/- S.D.) to Golgi (pH(G) = 6.2 +/- 0.4) to mature secretory granules (MSGs) (pH(MSG) = 5.5 +/- 0.4). Golgi and MSGs required active H(+) v-ATPases for acidification. ER, Golgi, and MSG steady-state pH values were also dependent upon the different H(+) leak rates across each membrane. However, neither steady-state pH(MSG) nor rates of passive H(+) leak were affected by Cl(-)-free solutions or valinomycin, indicating that MSG membrane potential was small and not a determinant of pH(MSG). Therefore, our data do not support earlier suggestions that organelle acidification is primarily regulated by Cl(-) conductances. Measurements of H(+) leak rates, buffer capacities, and estimates of surface areas and volumes of these organelles were applied to a mathematical model to determine the H(+) permeability (P(H+)) of each organelle membrane. We found that P(H+) decreased progressively from ER to Golgi to MSGs, and proper acidification of Golgi and MSGs required gradual decreases in P(H+) and successive increases in the active H(+) pump density.     

LNA for optimization of fluorescent oligonucleotide probes: improved spectral properties and target binding

Mixmer LNA/DNA fluorescent probes containing the 1-(phenylethynyl)pyrene fluorophore attached to 2'-arabino-uridine were synthesized and studied. The conjugates displayed significantly higher hybridization affinity to target DNA, increased fluorescence quantum yields of single-stranded oligonucleotides and their duplexes, and improved ability to form an interstrand excimer compared to analogous non-LNA probes.     

Sensitive immunoassay of Listeria monocytogenes with highly fluorescent bioconjugated silica nanoparticles probe

In this paper, a sensitive immunoassay method was proposed for Listeria monocytogenes detection by using highly fluorescent bioconjugated nanoparticles probe. (FITC-IgG)-doped fluorescent silica nanoparticles (fsNPs) firstly were synthesized by a microemulsion method and characterized by TEM and fluorescent spectra. Then the prepared fsNPs were conjugated with polyclonal rabbit anti-L. monocytogenes antibody (pAb) and used as indicator probe. A sandwich-type immune affinity reaction between polyclonal rabbit anti-L. monocytogenes antibody coated onto microplate wells, target bacteria and the fsNPs-antibody conjugates subsequently was conducted to detect target L. monocytogenes and assemble the indicator probe onto the wells. The target L. monocytogenes was measured by the fluorescent signals of the assembled indicator probes. Under the optimal conditions, the calibration graph of fluorescent intensity is proportional to the amount of target bacteria over the range of 50-10,320 CFU/mL with a detection limit of 50 CFU/mL. The proposed method has been successfully applied to detect L. monocytogenes in food samples offering the advantages of sensitivity, simplicity, and stability.     

Active electrogenic transport H+ in plasma membrane vesicles of cow parsnip phloem cells

Generation of electric (delta psi) and chemical (delta pH) components of electrochemical proton gradient delta muH+, in plasma membrane vesicles of Heracleum sosnovskyi phloem cells was investigated. ATP-dependent generation of delta psi at pH 6.0 in the presence of Mg2+ and K+ was established with the help of fluorescent probes AU+ and ANS-. Protonophore CCCP and proton ATPase inhibitor DCCD suppressed generation, whereas oligomycin, the inhibitor of mitochondrial ATPases did not affect it. Measurings of delta psi value indicated its oscillations within the limits from 10 to 60 mV. ATP-dependent generation of delta pH was established by means of fluorescent probe 9-AA. The effect was eliminated by CCCP and stimulated by K+, that may testify to the transformation of a part of delta psi into delta pH at antiport H+/K+. Existence of H+-ATPase in the plasma membranes of higher plant cells insuring generation of delta muH+ is supposed.     

Plasma membrane H+-ATPase is involved in auxin-mediated cell elongation during wheat embryo development

Previous investigations suggested that specific auxin spatial distribution due to auxin movements to particular embryonic regions was important for normal embryonic pattern formation. To gain information on the molecular mechanism(s) by which auxin acts to direct pattern formation in specific embryonic regions, the role of a plasma membrane (PM) ATPase was evaluated as downstream target of auxin in the present study. Western-blot analysis revealed that the PM H(+)-ATPase expression level was significantly increased by auxin in wheat (Triticum aestivum) embryos (two-three times increase). In bilaterally symmetrical embryos, the spatial expression pattern of the PM H(+)-ATPase correlates with the distribution pattern of the auxin analog, tritiated 5-azidoindole-3-acetic acid. A strong immunosignal was observed in the abaxial epidermis of the scutellum and in the epidermal cells at the distal tip of this organ. Pseudoratiometric analysis using a fluorescent pH indicator showed that the pH in the apoplast of the cells expressing the PM H(+)-ATPase was in average more acidic than the apoplastic pH of nonexpressing cells. Cellulose staining of living embryos revealed that cells of the scutellum abaxial epidermis expressing the ATPase were longer than the scutellum adaxial epidermal cells, where the protein was not expressed. Our data indicate that auxin activates the proton pump resulting in apoplastic acidification, a process contributing to cell wall loosening and elongation of the scutellum. Therefore, we suggest that the PM H(+)-ATPase is a component of the auxin-signaling cascade that may direct pattern formation in embryos.     

Golgi manganese transport is required for rapamycin signaling in Saccharomyces cerevisiae

The Pmr1 Golgi Ca2+/Mn2+ ATPase negatively regulates target of rapamycin complex (TORC1) signaling, the rapamycin-sensitive TOR complex in Saccharomyces cerevisiae. Since pmr1 causes resistance to rapamycin and tor1 causes hypersensitivity, we looked for genetic interactions of pmr1 with tor1. Deletion of TOR1 restored two wild-type phenotypes. Loss of TOR1 restored the ability of the pmr1 strain to grow on media containing 2 mm MnCl2 and conferred wild type as well as the wild-type sensitivity to rapamycin. Mn2+ additions to media partially suppressed rapamycin resistance of wild type and pmr1 tor1, suggesting that Tor1 and Tor2 are regulated by manganese. We parsed the roles of Ca2+ and Mn2+ transport and the compartments in rapamycin response using separation-of-function mutants available for Pmr1. A strain containing the D53A mutant (Mn2+ transporting) of Pmr1 is rapamycin sensitive, but the Q783A mutant (Ca2+ transporting) strain is rapamycin resistant. Mn2+ transport into the Golgi lumen appears to be required for rapamycin sensitivity. Overexpression of Ca2+ pump SERCA1, Ca2+/H+ antiporter Vcx1, or a Mn2+ transporting mutant of Vcx1 (Vcx1-M1) failed to restore rapamycin sensitivity, and loss of Pmr1 but not other transporters of Ca2+ or Mn2+ results in rapamycin resistance. Overexpression of Ccc1, a Fe2+ and Mn2+ transporter that has been localized to Golgi and the vacuole, does restore rapamycin sensitivity to pmr1Delta. We conclude that Mn2+ in the Golgi inhibits TORC1 signaling.     

The proton-translocating ATPase of Candida tropicalis plasma membrane

Proton translocation activity of Candida tropicalis plasma membrane ATPase has been demonstrated using a fluorescent delta pH probe (ACMA) and by direct pH measurements. Modifications in fluorescence intensity and H+ transport are highly specific for Mg2+ and ATP, and are sensitive to the well-known inhibitors of the plasma membrane ATPase, vanadate and DCCD. A H+/ATP ratio of 0.54 is found.     

Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of V-type ATPase from Arabidopsis

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.     

The pH dependence of the cardiac sarcolemmal Ca2(+)-transporting ATPase: evidence that the Ca2+ translocator bears a doubly negative charge

The pH dependence of the Ca2(+)-transporting ATPase of bovine cardiac sarcolemma was determined in a membrane vesicle preparation. The maximal velocity (Vmax) at saturating external Ca2+ showed a sigmoidal pH dependence with maximal values in the 6.0-6.5 range, a half-maximal value at 7.2 and minimal (less than or equal to 15%) values at pH greater than or equal to 8.0. The apparent affinity for Ca2+ (1/Km) varied over 10(4)-fold for 6.0 less than or equal to pH less than or equal to 8.5, increasing with increasing pH. Plots of log(1/Km) vs. pH were biphasic. In the acid range (6.0 less than or equal to pH less than or equal to 7.2), a slope of 2.6 was observed for the calmodulin-activated form of the pump. For 7.2 less than or equal to pH less than or equal to 8.5, a slope of 0.5 was observed. At pH 7.4, the Km is approx. 48 +/- 19 nM. The Ca2+ pump of cardiac sarcoplasmic reticulum in the same preparation had a Km of 304 +/- 115 nM and showed a similar pH dependence except that the slope in the acid range was 1.7. When calmodulin was removed from the sarcolemmal pump, its Km was raised to approx. 1.0 microM, the slope in the acid range was reduced to 1.7 and the Vmax was markedly reduced. The results are explicable in terms of a model in which each of the two Ca2+ binding sites on the pump contains two buried COO- groups responsible for high affinity. The Km effect is explained by 2 H+ vs. 1 Ca2+ competition for occupation of each of the two cytoplasmically-oriented translocators (4 H+ vs. 2 Ca2+). The Vmax effect is explained by counter-transport of H+. The findings are considered in terms of the published amino acid sequence of the cardiac sarcolemmal pump and recent site-directed mutagenesis vs. function studies identifying the Ca2+ binding site in the skeletal sarcoplasmic reticulum pump. The kinetic data are also applied to pump behavior under conditions of ischemia and acidosis.     

Structural and thermodynamic analysis of the GFP:GFP-nanobody complex

The green fluorescent protein (GFP)-nanobody is a single-chain VHH antibody domain developed with specific binding activity against GFP and is emerging as a powerful tool for isolation and cellular engineering of fluorescent protein fusions in many different fields of biological research. Using X-ray crystallography and isothermal titration calorimetry, we determine the molecular details of GFP:GFP-nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding. Although the GFP-nanobody can also bind YFP, it cannot bind the closely related CFP or other fluorescent proteins from the mFruit series. CFP differs from GFP only within the central chromophore and at one surface amino acid position, which lies in the binding interface. Using this information, we have engineered a CFP variant (I146N) that is also able to bind the GFP-nanobody with high affinity, thus extending the toolbox of genetically encoded fluorescent probes that can be isolated using the GFP-nanobody.     

Improving the sensitivity and dynamic range of reagentless fluorescent immunosensors by knowledge-based design

The variable fragment (Fv) of an antibody can be transformed into a reagentless fluorescent biosensor by mutating a residue into a cysteine in the neighborhood of the paratope (antigen-binding site) and then coupling an environment-sensitive fluorophore, e.g., N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester), to the mutant cysteine. For some residues, named operational, the formation of the conjugate does not affect the affinity of the Fv fragment for the antigen, and the binding of the antigen generates a measurable variation in the fluorescence intensity of the conjugate. We tested if this signal variation could be increased by coupling several molecules of fluorophores to the same molecule of Fv. Seven operational residues have been previously identified in the single-chain Fv (scFv) of monoclonal antibody D1.3 (mAbD1.3), directed against lysozyme. Ten double mutants of scFvD1.3, involving these residues, were constructed and coupled to the IANBD ester. The fluorescence of the double conjugates revealed a transfer of resonance energy between the two identical fluorescent groups. This homotranfer could be more important in the free state of the conjugate than in its antigen-bound state and increase its sensitivity for the detection of the antigen by up to 2.9-fold. A poorly sensitive conjugate could be improved by coupling a second molecule of fluorophore to residues located far from the paratope. Mutations altering the affinity of scFvD1.3 for lysozyme were introduced into one of its fluorescent conjugates. Using a mixture of three mutant derivatives of this unique conjugate, we could titrate lysozyme with precision in a concentration range encompassing 3 orders of magnitude.