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The structure of spinach thylakoid membranes has been investigated by sensitive differential scanning calorimetry. Six endotherms are observed between 20 and 85 degrees C, corresponding to order--disorder transitions of different structural domains within the thylakoid membrane. In a medium of relatively high ionic strength, endothermic transitions occur at 42, 54, 65, 72, 79, and 84 degrees C, with the 65 degrees C transition being particularly prominent. At a lower ionic strength, transitions are centered at 44, 61, 66, 70, 78, and 83 degrees C. The 42--44 degrees C endothermic transition (the A transition) can be correlated with the modification of three electron-transport components or properties associated with photosystem II: (i) release of manganese from the membrane, (ii) the loss of O2 evolution with water as a donor, and (iii) a decrease in the redox potential of the hydroquinone-reducible cytochrome b-559. Both the A transition and the ability to evolve O2 are irreversibly lost after heating to 49 degrees C and also after exposure to trypsin, suggesting the involvement of protein in this transition. The interpretation of these observations is that one effect of the A transition involves the thermal disruption of a protein component on the donor side of photosystem II.

The in vitro acidic pH dependence of colicin E1 channel activity was investigated by directed mutagenesis of Glu-468 in the colicin E1 channel domain, a residue conserved in the sequences of the four channel-forming colicins examined so far. Mutations were made to the amino acids leucine, serine, glutamine, or lysine, residues of different polarity and charge. All of the mutant polypeptides possessed high cytotoxic activity in vivo, although in vitro activity, especially with planar membranes, was lower than that of the wild type protein. A change in the in vitro acidic pH dependence of activity could be readily detected in the mutation to the hydrophobic leucine residue. The dependence of mutant activity on pH in the interval 3.5-5.0 was markedly smaller than that of the wild type, whether assayed on membrane vesicles or membrane bilayers. Differences in pH dependence between the wild type and the polar serine and glutamine mutants were small or of marginal statistical significance. No change in pH dependence could be detected with the charged lysine mutant. The residual pH dependence in all cases indicated that more than one carboxylic residue must be protonated to account for the increased activity at acidic pH values. A role of Glu-468 in the mechanism of channel formation or function was implied by the changes determined in vitro of channel parameters relative to the wild type: (i) the relatively small rates of current increase measured for colicin COOH-terminal peptide derived from the mutants, (ii) the small values of steady-state conductance of mutant peptide at pH 3.5, and (iii) the reduced anion selectivity of peptide from the serine mutant.

Colicin E1 was altered by oligonucleotide-directed mutagenesis at the site of three charged residues on the COOH side of the 35-residue hydrophobic segment in the channel-forming domain. Asp-509 is one of five conserved acidic residues in the channel domain of colicins A, B, E1, Ia, and Ib and is the first charged residue following the hydrophobic segment, followed by the basic residues Lys-510 and Lys-512. Asp-509 and Lys-512 were changed to amber and ochre stop codons, respectively, while Lys-510 was mutated to a Met codon. Proteins truncated after residue 508 or 511, and missing the last 14 or 11 residues, were obtained from a nonsuppressing cell strain harboring the mutant plasmid while full-length colicin molecules with single residue changes at Asp-509 to Leu, Ser, and Gln, and Lys-512 to Tyr, were obtained by using appropriate suppressor strains. The truncated colicins displayed (i) a low cytotoxicity, approximately 1% of intact wild-type colicin, (ii) 10-fold less in vitro channel activity with liposomes, and (iii) reduced labeling of the colicin in liposomes by a phospholipid photoaffinity probe, showing that one or more of the residues following Asn-511 is necessary for both in vivo and in vitro activity and insertion into the bilayer. (iv) The truncated mutants also displayed an altered conformation at pH 6 that allowed greater binding and activity with liposomes at this pH relative to wild type. The cytotoxicity of single residue substitutions at Asp-509 showed a range of cytotoxicities, wild type greater than Ser-509 greater than Gln-509 greater than Leu-509, although none of these changes greatly affected the in vitro channel activity or pH dependence.(ABSTRACT TRUNCATED AT 250 WORDS)

The secondary structure content of the COOH-terminal tryptic peptide of colicin E1 has been measured by analysis of UV circular dichroism spectra as a function of pH in aqueous medium and in the presence of the nonionic detergents octyl glucoside and Triton X-100. The alpha-helical content of the peptide increased by approximately 10%, from 45-47% to 56-57%, in the presence of the nonionic detergents, but not in aqueous medium, as the pH was decreased from 4.5 to 3.5. This pH dependence of conformation is similar to that reported elsewhere for the in vitro activity and binding of this peptide. A smaller increase in helical content was observed for the peptide in aqueous medium or in Triton X-100 as the pH was decreased from 6.5 to 4.5. The letter change in helical content was not seen in octyl glucoside which was present at a detergent:peptide stoichiometry 100 times that of Triton. The mean residue ellipticity measured at 222 nm for peptide added to asolectin vesicles by a freeze-thaw treatment was slightly larger at pH 3.5, and substantially larger at pH 4.5, than found at these pH values in the detergent solutions. Changes in helical content at the former, but not the latter pH, could be attributed to peptide insertion. It appears that protonation of one or more acidic amino acid residues in the COOH-terminal region of the molecule causes a conformational change that can be attributed to an extra helical domain that is stabilized in a nonpolar environment. From the similar pH dependence of the conformational change and in vitro binding and activity, it is inferred that interaction of this domain with the membrane is essential for binding and insertion.

In the presence of 0.1-5 muM N-methylphenazonium methosulphate approx. 50-70% oxidation of cytochrome b-559 can be induced by far-red light. The oxidation is best observed with long wavelength far-red light (732 nm) of moderate intensities (approx. 10(4) ergs/cm2 per s) and is reversed by subsequent illumination with red light. Concentrations of N-methylphenazonium methosulphate above 5 muM are inhibitory probably due to cyclic electron flow. The far-red oxidation is inhibited by low concentrations of the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, while 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibits red light reduction and increases the amplitude of far-red oxidation. The effect of N-methylphenazonium methosulphate is mimicked by N-methyl-phenazonium ethosulphate, but not by pyocyanine or diaminodurene. Low concentrations (2-3 muM) of N-methylphenazonium methosulphate also stimulate a 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-inhibitable red light reduction of cytochrome f.

Protein carboxyl methyltransferases, which catalyze transfer of methyl groups from S-adenosyl-L-methionine to the free carboxyl groups of acidic amino acids in proteins, can be divided into two classes based on several characteristics, such as the stoichiometry of substrate protein methylation, base stability of the incorporated methyl group, specificity for substrate, and participation in a regulatory system with which methylesterases are associated. The presence of such an enzyme in a photosynthetic system was demonstrated in the present work. The extent of methylation of chloroplast proteins was stimulated 30% by light and then decreased by the same amount in the presence of the electron transport inhibitor 3-(3',4'-dichlorophenyl)-1', 1'-dimethylurea or uncouplers of phosphorylation, indicating a dependence of the methyltransferase activity on photosynthetic electron transport and the trans-membrane delta pH. The light-independent, as well as the light-dependent, activity is probably of chloroplast origin since the extent of light stimulation in the purified thylakoid membranes and the stromal fraction was similar, and at low concentrations of S-adenosyl-L-methionine the small subunit of ribulose-1,5-bisphosphate carboxylase:oxygenase was found to be the predominant substrate. The labeling pattern of chloroplast proteins and labeling of an exogenous nonchloroplast protein indicated that the methyltransferase activity was not substrate-specific, although at low concentrations of the methyl donor, the small subunit of ribulose-1,5-bisphosphate carboxylase:oxygenase was labeled almost exclusively. Based on the low stoichiometry (less than 100 pmol/mg protein) of the methylation, its base lability, irreversibility, and the lack of substrate specificity except at very low concentrations of methyl donor, it was inferred that the chloroplast methyltransferase is best classified as a class II system that may function as part of a repair mechanism to replace racemized amino acids.

Optical, resonance Raman, and electron paramagnetic resonance spectroscopies have been used to characterize the ligands and spin state of the chloroplast cytochrome b-559. The protein was isolated from both maize and spinach in a low-potential form. The spectroscopic data indicate that the heme iron in both ferric and ferrous cytochrome b-559 is in its low-spin state and ligated in its fifth and sixth coordination positions by histidine nitrogens. Electron paramagnetic resonance data for the purified spinach cytochrome are in good agreement with those determined by Bergström and Vänngård [Bergström, J., & Vänngård, T. (1982) Biochim. Biophys. Acta 682, 452-456] for a low-potential membrane-bound form of cytochrome b-559. The g values of high-potential cytochrome b-559 are shifted from those of its low-potential forms; this shift is interpreted as arising from a deviation of the planes of the two axial histidine imidazole rings from a parallel orientation. The model is consistent with the physical data and may also account for the facility with which cytochrome b-559 can be converted between low- and high-potential forms. Recent biochemical and molecular biological data [Widger, W. R., Cramer, W. A., Hermodson, M., Meyer, D., & Gullifor, M. (1984) J. Biol. Chem. 259, 3870-3876; Herrmann, R. G., Alt, J., Schiller, D., Cramer, W. A., & Widger, W. R. (1984) FEBS Lett. 179, 239-244] have shown that two polypeptides, one with 83 residues and a second with 39 residues, most likely constitute the protein of the cytochrome.(ABSTRACT TRUNCATED AT 250 WORDS)

The dependence of colicin channel activity on membrane potential and peptide concentration was studied in large unilamellar vesicles using colicin E1, its COOH-terminal thermolytic peptide and other channel-forming colicins. Channel activity was assayed by release of vesicle-entrapped chloride, and could be detected at a peptide: lipid molar ratio as low as 10(-7). The channel activity was dependent on the magnitude of a transnegative potassium diffusion potential, with larger potentials yielding faster rates of solute efflux. For membrane potentials greater than -60 mV (K+in/K+out greater than or equal to 10), addition of valinomycin resulted in a 10-fold increase in the rate of Cl- efflux. A delay in Cl-efflux observed when the peptide was added to vesicles in the presence of a membrane potential implied a potential-independent binding-insertion mechanism. The initial rate of Cl- efflux was about 1% of the single-channel conductance, implying that only a small fraction of channels were initially open, due to the delay or latency of channel formation known to occur in planar bilayers. The amount of Cl- released as a function of added peptide increased monotonically to a concentration of 0.7 ng peptide/ml, corresponding to release of 75% of the entrapped chloride. It was estimated from this high activity and consideration of vesicle number that 50-100% of the peptide molecules were active. The dependence of the initial rate of Cl- efflux on peptide concentration was linear to approximately the same concentration, implying that the active channel consists of a monomeric unit.

The region of the colicin E1 polypeptide that interacts with immunity protein has been localized to a 168-residue COOH-terminal peptide. This is the length of a proteolytically generated peptide fragment of colicin E1 against which imm+ function can be demonstrated in osmotically shocked cells. The role of particular amino acids of the COOH-terminal peptide in the expression of the immune phenotype was studied. Chemical modification showed that the two histidine residues (His 427 and His 440) and the single cysteine residue (Cys 505) present in the COOH-terminal peptide were not necessary for the colicin-immunity protein interaction. The immunity protein was localized in the cytoplasmic membrane fraction, consistent with previous work of others on the colicin Ia immunity protein and the prediction from the immunity protein amino acid sequence that it is a hydrophobic protein. The distribution of hydrophobic residues along the immunity polypeptide was calculated.

The topography of chloroplast cytochromes f and b6 was probed with proteases carboxypeptidase A (CpA), trypsin, and Staph, aureus V8. The cytochrome and its proteolytic products were detected by heme stain and, in most experiments, by immunoreaction. In thylakoids, the only protease that significantly affected the intactness of cytochrome f was CpA that caused a small (delta Mr = -1-2000) decrease in the apparent molecular weight. In SDS-treated thylakoids, both trypsin and V8 degraded cytochrome f. The inferred topography of cytochrome f., with the COOH-terminus on the stromal (n) side, one membrane-spanning alpha-elix near the COOH-terminus, and most of the Cyt f mass on the lumen (p) side, is consistent with that previously inferred by others. Cytochrome b6 was not sensitive to CpA, but was more sensitive to trypsin and V8 protease than cytochrome f, cytochrome b-559, or the 17 kDa OEC extrinsic protein. Trypsin caused a small decrease in size of cytochrome b6, which was observed using whole protein antibody as a single smaller band (delta Mr approximately 2000) or two smaller discrete bands (delta Mr = -1000 and 2500, respectively) which, unlike the untreated protein, did not react with antibody generated to a peptide mimicking Asp-5-Gln-14 near the NH2-terminus. These shortened tryptic fragments were attributed to cleavage after R-10 and K-23 near the NH2-terminus, implying an orientation with the NH2-terminus on the stromal side of the membrane. The sensitivity of cytochrome b6 toward this trypsin cleavage was increased if the membranes were first incubated with CpA, showing that the NH2-terminal region of cytochrome b6 is masked by the COOH-terminal domain of one or more thylakoid proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

The fluorescent probe, 8-anilino-1-napthalenesulfonate (ANS) binds to Escherichia coli, showing an enhanced fluorescence. The interaction of colicin E1 with sensitive cells causes an increase of about 100% in the fluorescence of the bound ANS, and this change at equilibrium has an apparent "all-or-none" nature as a function of E1 multiplicity. Approximately 6 to 8% of the ANS is bound to the cells at equilibrium. The colicin E1-induced fluorescence increase can be attributed partly to an increase in ANS binding and partly to an increase in the fluorescence yield of the bound ANS. The kinetics of the E1-induced fluorescence increase in sensitive cells are very similar to those of the adenosine triphosphate decrease. The phosphorylation uncoupler p-trifluoromethoxy-carbonylcyanidephenylhydrazone also causes a large change in the fluorescence of bound ANS. Colicin E2 or E3 does not cause any fluorescence change, nor does colicin E1 cause fluorescence change with a colicinogenic strain. ANS appears to be a probe of structural or conformational change in the cell envelope that is closely associated with the colicin E1-induced adenosine triphosphate decrease.