Eosin Isothiocyanate Research Articles

Evidence for a sequestered solvent space in the chloroplast ATPase

Isolated coupling factor of photophosphorylation (CF 1) covalently labeled with eosin isothiocyanate was studied by polarized laser spectroscopy. Judged by the access of oxygen bound to eosin isothiocyanate and by the librational mobility of eosin isothiocyanate we conclude that activated CF 1 encloses a volume with solvent character. In the membrane-bound enzyme the sequestered volume becomes exposed when the membrane is energized.

On the conformation of reconstituted ferredoxin:NADP + oxidoreductase in the thylakoid membrane. Studies via triplet lifetime and rotational diffusion with eosin isothiocyanate as label

Eosin isothiocyanate was covalently bound to isolated ferredoxin-NADP + reductase under protection of the NADP-binding domain. The bound label did not impair the functional reconstitution of the enzyme into depleted thylakoid membranes. Laser spectrophotometric experiments were carried out on thylakoids which were reconstituted with labeled ferredoxin-NADP + reductase. Bound eosin isothiocyanate was used as a spectroscopic probe for conformational changes of ferredoxin-NADP + reductase in either of two ways: We studied the rotational diffusion of labeled ferredoxin-NADP + reductase in the membrane by the photoselection technique, and we studied the triplet lifetime of bound eosin, which measures polypeptide chain flexibility (via access of oxygen) around the binding site. The latter technique was complemented by measurements of the librational motion of bound dye. We observed: (1) When ferredoxin is absent, ferredoxin-NADP + reductase undergoes very rapid rotational diffusion in the thylakoid membrane (correlation time less than 1 μs at 10°C). This is drastically slowed down (40 μs) upon addition of water-soluble ferredoxin. We propose that ferredoxin mediates the formation of a ternary complex with ferredoxin-NADP + reductase and the Photosystem I complex. According to our data, this complex would live longer than required for the photoreduction of ferredoxin-NADP + reductase by Photosystem I via ferredoxin. (2) Under the given incubation conditions, the binding sites for eosin isothiocyanate were located in the FAD domain of ferredoxin-NADP + reductase. We found increased chain flexibility in this domain upon addition of NADP. This suggests induced fit for the binding of NADP and allosteric control of the FAD domain by the remote NADP domain. (3) Acidification of the internal phase of thylakoids decreased the chain flexibility in the FAD domain. This is of particular interest, since ferredoxin-NADP + reductase is a peripheral external membrane protein. It suggests the existence of a binding protein for the oxidoreductase which spans the membrane and senses the internal pH

Coupling factor for photophosphorylation labeled with eosin isothiocyanate: activity, size, and shape in solution.

The coupling factor for photophosphorylation (CF1) was covalently labeled with eosin isothiocyanate (ESCN). We found extra binding sites for the label when a protonmotive force existed across the thylakoid membrane during incubation with ESCN. Two out of three such extra sites are located on the y subunit of CF1. As judged from the (oxygen-dependent) triplet lifetime of bound ESCN, these extra sites are more deeply buried within CF1 than those sites which are already accessible to ESCN in the absence of a protonmotive force. Labeling of the extra sites by only one ESCN per CF1 greatly reduced the activities of the enzyme (more strongly ATP synthesis and Mgz+-dependent hydrolysis of the membrane-bound CFl than the Caz+-dependent hydrolysis). On the other hand, a load of up to five ESCN's when bound in the absence of a protonmotive force had hardly Synthesis of ATP in green plants is mediated by the coupling factor (CFl).' This enzyme is composed from five types of subunits, and it is bound to the thylakoid membrane via its counterpart (CFO), which acts as a proton well [for recent reviews, see Nelson (1976), Kagawa et al. (1979), and Shavit (1980)l. It seems generally accepted that these two together act as a proton-translocating ATP synthase as proposed by Mitchell (1966). In comparison with the ATP synthase complexes of bacteria and of mitochondria, the one of green plants is distinguished by its apparent irreversibility in the From the Schwerpunkt Biophysik, Fachbereich Biologie/Chemie, Universitat Osnabrlick, D-4500 Osnabriick, West Germany. Received June 17,1981. Financially supported by Niedersachsisches Ministerium fiir Wissenschaft and Knnst (VW-Vorab) and by Deutsche Forschungsgemeinschaft. 0006-2960 /82 /04211890SO 1.25 /O any effect. We isolated and purified active CF1 which was labeled in the absence of a protonmotive force. We studied the rotational diffusion of the isolated enzyme by a photoselection technique aiming at the triplet state of bound eosin. A biphasic decay of the photoinduced linear dichroism of the absorption changes of eosin was observed. The more rapid component was only slightly dependent on the solvent viscosity and therefore attributed to librational motion of the label within the protein. The decay of the slower component was linearly related to the solvent viscosity and therefore attributed to protein rotational diffusion. The theoretical evaluation of the data led us to conclude that isolated CF1 is ellipsoidal rather than spherical in shape, with an axial ratio which is greater than 2.1 if it is prolate and smaller than 1/2.6 if it is oblate. absence of a protonmotive force (Petrack & Lipman, 1961; Kaplan et al., 1967; Mills & Hind, 1979). It is activated under conditions which are also known to induce large conformational changes of CF1 when a protonmotive force is generated by illumination, by an acid-base jump, or by externally applied electric field pulses (Ryrie & Jagendorf, 1971; McCarty & Fagan, 1973; Graeber et al., 1977; Weiss & McCarty, 1977; Kraayenhof & Slater, 1975). The same conditions promote the release (or the exchange) of tightly bound nucleotides ' Abbreviations: CFI , coupling factor for photophosphorylation; ESCN, eosin isothiocyanare; 5-IAF, 5-(iodoacetamido)fluorescein; DTT, dithiothreitol; NBD-CI, 7-chloro-4-nitro-2,1,3-benzoxadiazole; Tricine, N-[tris(hydroxymethyI)methyl]glycine; NaDodSO,, sodium dodecyl sulfate; EDTA, ethylenediaminetetraacetic acid; fwhm. full width at half-maximum.

Chemical modification of matrix porin from Escherichia coli: probing the pore topology of a transmembrane protein.

Chemical modification of amino groups in matrix porin solubilized and purified from outer membranes of Escherichia coli in beta-octylglucoside was performed with eosin isothiocyanate and citraconic anhydride. At pH 7 8.5, the former reagent labeled a single amino group in the native protein, while more extensive derivatization was observed with increasing pH or upon denaturation. Citraconic anhydride modified approximately 12-14 residues in native porin and 15-16 of the total of 19 amino groups in the denatured state. Fluorescamine, another amine-specific reagent of intermediate size, derivatized 3 and 16 residues in the native and denatured states, respectively. These results indicate that reactive probes of various sizes may serve as indicators for the surface accessibility of reactive residues in matrix porin. The increased derivatization of lysyl residues at high pH (or in phosphate buffer) suggests the method's sensitivity to different conformational states of the protein. The extent of tyrosine modification (1-2 residues in the native, and approximately 22 in the denatured porin) depended on the state of protein folding, even with reagents of small size. The approach of using various probes with differing properties and specificities thus appears useful for the determination of membrane protein asymmetry, pore topology, and conformational states of transmembrane proteins.

Protein rotation, enzyme activity and photooxidation of SH groups in sarcoplasmic reticulum Ca 2+-ATPase

The binding of probe molecules such as fluorescein isothiocyanate, eosin isothiocyanate and erythrosin isothiocyanate to the Ca 2+-ATPase of sarcoplasmic reticulum followed by illumination of the labelled protein causes substantial reductions of ATPase activity over a 1-h period. The degree of light-sensitivity induced by these probes is related to the triplet yield of these probe molecules. Consistent with this, the greatest effect is seen with erythrosin isothiocyanate and the least effect with fluorescein isothiocyanate. These reductions of ATPase activity associated with illumination are also associated with an aggregation of the protein molecules. This is indicated by laser flash photolysis measurements and also by polyacrylamide gel electrophoresis. A reduction in the number of thiol groups present on the ATPase molecule parallels the reduction of enzyme activity and changes in the protein mobility. The results are discussed in relation to the use of these probe molecules to study biological systems and also in terms of oxidative processes which may affect protein function in vivo.

Lateral mobility of band 3 in the human erythrocyte membrane studied by fluorescence photobleaching recovery: evidence for control by cytoskeletal interactions.

Band 3, the major intrinsic protein of the human erythrocyte membrane, was specifically labeled with the covalent fluorescent probe eosin isothiocyanate. The lateral mobility of labeled band 3 in the plane of the membrane under various conditions of ionic strength and temperature was examined by using the fluorescence photobleaching recovery technique. Low temperature (21 degrees C) and high ionic strength (46 mM NaPO(4)) favored immobilization of band 3(10% mobile) as well as slow diffusion of the mobile fraction (diffusion coefficient D = 4 x 10(-11) cm(2)sec(-1)). Increasing temperature (37 degrees C) and decreasing ionic strength (13 mM NaPO(4)) led to an increase in the fraction of mobile band 3(90% mobile) and a reversible increase in the diffusion rate of the mobile fraction (D = 200 x 10(-11) cm(2)sec(-1)). The increase in the fraction of mobile band 3 was markedly dissociated, however, from the increase in the diffusion rate of the mobile fraction. Thus, the fraction of mobile band 3 always increased at higher ionic strength and lower temperature than the ionic strength and temperature at which the diffusion rate increased. This dissociation was manifested kinetically on prolonged incubation of ghosts at constant ionic strength and temperature: the diffusion rate of the mobile fraction increased slowly at first and much more rapidly after the initial lag period, whereas the fraction of mobile band 3 increased almost immediately to 90% and remained maximal for the duration of the experiment. Further, changes in diffusion rate with temperature were promptly and totally reversible, whereas increases in the mobile fraction were only slowly and partially reversible. These effects were shown not to be due to complete dissociation of spectrin, the major protein of the erythrocyte cytoskeleton, from the membrane. This evidence suggests control of band 3 lateral mobility by at least two separate processes. The process that determines the diffusion coefficient of the mobile band 3 is completely reversible, and it probably involves a metastable state of cytoskeleton structure intermediate between tight binding to the membrane and complete dissociation from it.

Rotational motion and evidence for oligomeric structures of sarcoplasmic reticulum Ca2+-activated ATPase.

The rotational motion of the sarcoplasmic reticulum Ca2+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) has been investigated by measuring the decay of laser flash-induced dichroism with the covalently attached triplet probe eosin isothiocyanate. The Arrhenius plot for rotational mobility indicates two discontinuities at approximately 15 degrees C and approximately 35 degrees C. The experimental data are rationalized in terms of a sudden conformeric change in the ATPase at 15 degrees C and a temperature-dependent equilibrium existing between the conformationally altered ATPase and oligomeric forms of it in the temperature range 15-35 degrees C. The enzymatic activity, as indicated by a discontinuity in the Arrhenius plot for the rate of ATP hydrolysis, appears to be sensitive only to the change at 15 degrees C. There is a strong correlation between the activation energy below 15 degrees C for rotational motion (33.6 +/- 2.2 kcal/mol) and enzymatic activity (34 +/- 4 kcal/mol).