Development of a fluorescent probe for the study of nucleosome assembly and dynamics.

To develop a probe for use in real-time dynamic studies of nucleosomes, core histones (from Drosophila) were conjugated to a DNA-intercalating dye, thiazole orange, by a reaction targeting Cys 110 of histone H3. In the absence of DNA, the conjugated histones are only very weakly fluorescent. However, upon reconstitution into nucleosomes by standard salt dialysis procedures, the probe fluoresces strongly, reflecting its ability to intercalate into the nucleosomal DNA. The probe is also sensitive to the nature of the DNA-histone interaction. Nucleosomes reconstituted by stepwise salt dialysis give a fluorescence signal quite different from that of the species formed when DNA and histones are simply mixed in low salt. In addition, changing either the DNA length or the type of sequence (nucleosome positioning sequences versus random DNA of the same size) used in the reconstitution alters the resulting fluorescence yield. The results are all consistent with the conclusion that a more rigid, less flexible nucleosome structure results in less fluorescence than a looser structure, presumably due to structural constraints on dye intercalation. This probe should be well suited to analyzing nucleosome dynamics and to following factor-mediated assembly and remodeling of nucleosomes in real time, particularly at the single-molecule level.

Blue light drives B-side electron transfer in bacterial photosynthetic reaction centers.

The core of the photosynthetic reaction center from the purple non-sulfur bacterium Rhodobacter sphaeroides is a quasi-symmetric heterodimer, providing two potential pathways for transmembrane electron transfer. Past measurements have demonstrated that only one of the two pathways (the A-side) is used to any significant extent upon excitation with red or near-infrared light. Here, it is shown that excitation with blue light into the Soret band of the reaction center gives rise to electron transfer along the alternate or B-side pathway, resulting in a charge-separated state involving the anion of the B-side bacteriopheophytin. This electron transfer is much faster than normal A-side transfer, apparently occurring within a few hundred femtoseconds. At low temperatures, the B-side charge-separated state is stable for at least 1 ns, but at room temperature, the B-side bacteriopheophytin anion is short-lived, decaying within approximately 15 ps. One possible physiological role for B-side electron transfer is photoprotection, rapidly quenching higher excited states of the reaction center.

Characterization of a Rhodobacter capsulatus reaction center mutant that enhances the distinction between spectral forms of the initial electron donor.

A large scale mutation of the Rhodobacter capsulatus reaction center M-subunit gene, sym2-1, has been constructed in which amino acid residues M205-M210 have been changed to the corresponding L subunit amino acids. Two interconvertable spectral forms of the initial electron donor are observed in isolated reaction centers from this mutant. Which conformation dominates depends on ionic strength, the nature of the detergent used, and the temperature. Reaction centers from this mutant have a ground-state absorbance spectrum that is very similar to wild-type when measured immediately after purification in the presence of high salt. However, upon subsequent dialysis against a low ionic strength buffer or the addition of positively charged detergents, the near-infrared spectral band of P (the initial electron donor) in sym2-1 reaction centers is shifted by over 30 nm to the blue, from 852 to 820 nm. Systematically varying either the ionic strength or the amount of charged detergent reveals an isobestic point in the absorbance spectrum at 845 nm. The wild-type spectrum also shifts with ionic strength or detergent with an isobestic point at 860 nm. The large spectral separation between the two dominant conformational forms of the sym2-1 reaction center makes detailed measurements of each state possible. Both of the spectral forms of P bleach in the presence of light. Electrochemical measurements of the P/P+ midpoint potential of sym2-1 reaction centers show an increase of about 30 mV upon conversion from the long-wavelength form to the short-wavelength form of the mutant. The rate constant of initial electron transfer in both forms of the mutant reaction centers is essentially the same, suggesting that the spectral characteristics of P are not critical for charge separation. The short-wavelength form of P in this mutant also converts to the long-wavelength form as a function of temperature between room temperature and 130 K, again giving rise to an isobestic point, in this case at 838 nm for the mutant. A similar, though considerably less pronounced spectral change with temperature occurs in wild-type reaction centers, with an isobestic point at about 855 nm, close to that found by titrating with ionic strength or detergent. Fitting the temperature dependence of the sym2-1 reaction center spectrum to a thermodynamic model resulted in a value for the enthalpy of the conformational interconversion between the short- and long-wavelength forms of about -6 kJ/mol and an entropy of interconversion of about -35 J/(K mol). Similar values of enthapy and entropy changes can be used to model the temperature dependence in wild-type. Thus, much of the temperature dependence of the reaction center special pair near-infrared absorbance band can be described as an equilibrium shift between two spectrally distinct conformations of the reaction center.

P+HA- charge recombination reaction rate constant in Rhodobacter sphaeroides reaction centers is independent of the P/P+ midpoint potential.

The kinetics of the P+HA- (oxidized donor, reduced bacteriopheophytin acceptor) recombination reaction was measured in a series of reaction center mutants of Rhodobacter sphaeroides with altered P/P+ midpoint potentials between 410 and 765 mV. The time constant for P+HA- recombination was found to range between 14 and 26 ns and was essentially independent of P/P+ midpoint potential. Previous work has shown that the time constant for initial electron transfer in these mutants at room temperature is also only weakly dependent on the P/P+ midpoint potential, ranging from about 2.5 ps to about 50 ps. These results, taken together, imply that heterogeneity in the P/P+ midpoint potential within the reaction center population is not likely the dominant cause of the substantial kinetic complexity observed in the decay of the excited singlet state of P on the picosecond to nanosecond time scale. In addition, the pathway of P+HA- decay appears to be direct or via P+BA- rather than proceeding back through P, even in the highest-potential mutant, as is evident from the fact that the rate of P+HA- recombination is unaltered by pushing P+HA- much closer to P in energy. Finally, the midpoint potential independence of the P+HA- recombination rate constant suggests that the slow rate of P+HA- recombination arises from an inherent limitation in the maximum rate of this process rather than because it occurs in the inverted region of a classical Marcus rate vs free energy curve.

Asymmetry requirements in the photosynthetic reaction center of Rhodobacter capsulatus.

Nine large-scale symmetry reaction center mutants were constructed in Rhodobacter capsulatus by replacing segments of the M subunit gene with the homologous region of the L subunit gene. Between them, the mutations resulted in symmetrization of essentially the entire region from the carboxy terminal portion of the C helix through most of the E helix. The amino acids in this region define about 80% of the environment of the reaction center cofactors. These studies show that roughly 80% of the amino acids that come in close contact with the cofactors involved in initial electron transfer can be made symmetric in a piecewise manner without loss of the ability to grow photoheterotrophically. However, the amino acid regions near the quinones and iron atom are much more sensitive to symmetrization and most of the large-scale changes in this region resulted in the loss of photosynthetic viability, probably due to loss of stable reaction centers from the photosynthetic membrane. More detailed analysis of the isolated photosynthetic membranes from these mutants showed that in all cases but one, there was some amount of charge separation occurring in the mutant reaction centers. This bank of mutants serves as a useful starting point for more detailed studies of the differential molecular interactions which occur between the two reaction center subunits and their associated cofactors.

Low-temperature femtosecond-resolution transient absorption spectroscopy of large-scale symmetry mutants of bacterial reaction centers.

Reaction centers isolated from three large-scale symmetry mutants sym0, sym2-1, and sym5-2 described in the previous article of this issue [Taguchi, A. K. W., Eastman, J. E., Gallo, D. M., Jr., Sheagley, E.. Xiao, W., & Woodbury, N. W. (1996) Biochemistry 35, 3175-3186] have been investigated by low-temperature ground state and ferntosecond-resolution transient absorption spectroscopy. All three of these large-scale symmetry mutants undergo electron transfer at 20 K. The mutants sym0 and sym5-2 have yields and dominant rates of charge separation comparable to wild type. However. the sym2-mutant shows a roughly 35%, quantum yield at this temperature, and the major kinetic component of the initial electron transfer is slower than wild type by nearly a factor of 100. The sym0 mutant showed substantial changes in the monomer bacteriochiorophyll ground state and transient spectra, and both sym0 sym2-1 showed changes in the bacteriopheophyll ground state and transient spectra. In particular, sym2-1 shows a small absorbance decrease in the region of the Qx band of the B side bacteriopheophytin which could be attributed to 10%-20% electron transfer along the B pathway.

Time-dependent thermodynamics during early electron transfer in reaction centers from Rhodobacter sphaeroides.

The temperature dependence of fluorescence on the picosecond to nanosecond time scale from the reaction centers of Rhodobacter sphaeroides strain R-26 and two mutants with elevated P/P+ midpoint potentials has been measured with picosecond time resolution. In all three samples, the kinetics of the fluorescence decay is complex and can only be well described with four or more exponential decay terms spanning the picosecond to nanosecond time range. Multiexponential fits are needed at all temperatures between 295 and 20 K. The complex decay kinetics are explained in terms of a dynamic solvation model in which the charge-separated state is stabilized after formation by protein conformational changes. Many of these motions have not had time to occur on the time scale of initial electron transfer and/or are frozen out at low temperature. This results in a time- and temperature-dependent enthalpy change between the excited singlet state and the charge-separated state that is the dominant term in the free energy difference between these states. Long-lived fluorescence is still observed even at 20 K, particularly for the high-potential mutants. This implies that the driving force for electron transfer on the nanosecond time scale at low temperature is less than 200 cm-1 (25 meV) in R-26 reaction centers and even smaller on the picosecond time scale or in the high-potential mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between thermodynamics and mechanism during photoinduced charge separation in reaction centers from Rhodobacter sphaeroides.

Detailed fast transient absorption measurements have been performed at low temperature on reaction centers from Rhodobacter sphaeroides strain R-26 and on a double mutant, [LH(L131) + LH-(M160)], in which the P/P+ oxidation potential is roughly 140 mV (1100 cm-1) above that of wild-type reaction centers. In both samples, the decay of the excited singlet state of the initial electron donor is not well described by a single-exponential decay term. This is particularly true for reaction centers from the double mutant where at least three exponential kinetic components are required to describe the decay, with time constants ranging from a few picoseconds to hundreds of picoseconds. However, singular value decomposition analysis of the time-dependent absorption change spectra indicates the presence of only two spectrally distinct states in reaction centers from both R-26 and the double mutant. Thus, the complex decay of P* at low temperature does not appear to be due to formation of either the state P+BA- as a distinct intermediate in electron transfer or P+BB- as an equilibrated side product of electron transfer. Instead, the decay kinetics are modeled by assuming dynamic solvation of the charge-separated state, as was done for the long-lived fluorescence decay in the accompanying paper [Peloquin, J. M., Williams, J. C., Lin, X., Alden, R. G., Taguchi, A. K. W., Allen, J.P., & Woodbury, N. W. (1994) Biochemistry 33, 8089-8100]. The results of assuming a static distribution of electron-transfer rates at early times followed by dynamic solvation of the charge-separated states on longer time scales are also presented. Regardless of which model is used to describe the early time kinetics of excited-state decay, the time-dependent excited-state population on the 100-ps or longer time scale is best described in terms of thermal repopulation of P* from the charge-separated state, even at 20 K. This results in a time- and temperature-dependent driving force estimated for initial electron transfer of less than 200 cm-1 on all time scales from picoseconds to nanoseconds. Assuming a nonzero internal reorganization energy associated with charge separation, the small driving force does not appear to be consistent with the lack of temperature dependence of electron transfer and the fact that a mutant with a P/P+ oxidation potential 140 mV (1100 cm-1) higher than wild type is still able to undergo electron transfer, even at low temperature.(ABSTRACT TRUNCATED AT 400 WORDS)

Femtosecond pump-probe analysis of energy and electron transfer in photosynthetic membranes of Rhodobacter capsulatus.

Low-intensity, 295 K, femtosecond pump-probe transient absorption measurements are described that have been performed to investigate energy and electron transfer in photosynthetic membranes from a Rhodobacter capsulatus strain lacking functional light harvesting antenna complex II. Spectral and kinetic similarities between the absorption changes of isolated reaction centers and those of reaction centers in membranes upon 800-nm excitation suggest that the charge separation process in both cases is very similar. An ultrafast energy relaxation process observed near 872 nm when 800-nm excitation is used is interpreted as interexcitonic relaxation within the antenna, though other interpretations, such as vibrational relaxation, are possible. On the basis of global exponential fitting analysis of the time-dependent spectral changes using 800- and 880-nm excitation wavelengths to selectively excite the reaction center and the LHI antenna, respectively, it is found that excitation energy transfer and trapping in Rb. capsulatus is limited by the overall rate of energy transfer between the antenna and the reaction center. This conclusion is supported by the observation that excitation at 800 nm, but not 880 nm, results in absorbance changes indicative of charge separation with a lifetime (3.1 ps) very close to that reported for charge separation in isolated reaction centers (3.5 ps). Thus, most reaction centers that are directly excited undergo charge separation and not backward energy transfer to the LHI antenna complexes. Both a kinetic model analysis and a direct comparison between time-resolved spectra obtained using different excitation wavelengths resulted in an energy-detrapping efficiency of about 15 +/- 10%.

Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131.

Structural changes in chromatophores of Rhodobacter sphaeroides reaction center mutants associated with the substitution of amino acid residues near the primary electron donor P have been investigated by light-induced FTIR difference spectroscopy. The single-site mutations Leu-L131 to His and Leu-M160 to His and the corresponding double mutation were designed to introduce a proton-donating residue that could form a hydrogen bond with the keto carbonyl of ring V of each bacteriochlorophyll (PL and PM) of the dimer. The presence of large positive bands at approximately 1550, 1480, and 1295 cm-1, as well as at 2600-2800 cm-1 in the light-induced P+QA-/PQA FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone QA, demonstrates that the BChl dimer state of P+ is preserved in the LH(L131), LH(M160), and LH(M160)+LH(L131) mutants, although frequency shifts and amplitude changes can be observed, notably for LH(M160). Compared to wild type, these changes are thought to reflect a different charge repartition over the two BChls in P+. Large frequency downshifts in the 9-keto C=O stretching region of the P+QA-/PQA FTIR difference spectra of chromatophores are observed in the mutant samples relative to wild type. For the LH(M160) mutant, a large differential signal at 1678/1664 cm-1 is assigned to a shift, upon photooxidation, of the 9-keto C=O of PM hydrogen-bonded to His-M160, while that at 1718/1696 cm-1 corresponds to the free 9-keto C=O of PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations designed to modify the environment of the primary electron donor of the reaction center from Rhodobacter sphaeroides: phenylalanine to leucine at L167 and histidine to phenylalanine at L168.

Two mutations, L168 His to Phe and L167 Phe to Leu, were made in residues near the primary electron donor, a bacteriochlorophyll dimer, of the reaction center from Rhodobacter sphaeroides. Blue shifts of 10-15 nm in the 865-nm band of the donor were observed in the optical absorption spectra of both of the mutant reaction centers. The rate of initial electron transfer was determined by measurement of the kinetics of the decay of the excited state of the donor, and the rate of charge recombination was determined by measurement of the recovery of the bleaching of the donor. The initial electron transfer time constant and the charge recombination time constant were determined to be 3.6 ps and 220 ms, respectively, in the L168 His to Phe mutant and 5.0 ps and 85 ms in the L167 Phe to Leu mutant, compared to 3.8 ps and 100 ms measured for the wild type. The oxidation potential of the donor measured by oxidation-reduction titrations was found to decrease by 80 mV in the L168 His to Phe mutant and increase by 25 mV in the L167 Phe to Leu mutant. Time-resolved fluorescence decay measurements indicated that the change in the oxidation potential of the donor in the L168 His to Phe mutant resulted in a change in the energies of the charge-separated states. The results show that an increase in the driving force does not increase the rate of the initial electron transfer reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides.

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3' of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3' end of pufM and sequences past the end of pufM.

Biochemical characterization and electron-transfer reactions of sym1, a Rhodobacter capsulatus reaction center symmetry mutant which affects the initial electron donor.

A 51 bp section of the Rhodobacter capsulatus photosynthetic reaction center M subunit gene (nucleotides M562-M612 of the pufM structural sequence) encoding amino acids M187-M203 was replaced by the homologous region of the L subunit gene. This resulted in the symmetrization of much of the amino acid environment of the reaction center initial electron donor, P. This is the first in a series of large-scale symmetry mutations and is referred to as sym1. The sym1 mutant was able to grow photosynthetically, indicating that reaction center function was largely intact. Isolated reaction centers showed an approximately 10-nm blue shift in the QY band of P. The standard free energy change between P* and P+BphA- determined from analysis of the long-lived fluorescence from quinone-reduced reaction centers decreased from about -120 meV in the wild-type to about -75 meV in the sym1 mutant. A 65-70% quantum yield of electron transfer from P* to P+QA- was observed, most of the yield loss occurring between P* and P+BphA-. The decay of the stimulated emission from P* was about 3-fold slower in this mutant than in the wild-type. Time-resolved spectral analysis of the charge-separated intermediates formed in sym1 reaction centers indicated that the major product was P+BphA-. A model-dependent analysis of the observed rates and electron-transfer yields gave the following microscopic rate constants for sym1 reaction centers (wild-type values under the same conditions are given in parentheses): [formula: see text] Analysis of the sym1 mutant, mutants near P made by other groups, and interspecies variation of amino acids in the vicinity of P suggests that the protein asymmetry in the environment of the initial electron donor is important for optimizing the rate and yield of electron transfer, but is not strictly required for overall reaction center function.

Spectroscopic and redox properties of sym1 and (M)F195H: Rhodobacter capsulatus reaction center symmetry mutants which affect the initial electron donor.

The redox properties, absorption, electroabsorption, CD, EPR, and P+QA- recombination kinetics have been measured for the special pairs of two mutants of Rhodobacter capsulatus reaction centers involving amino acid changes in the vicinity of the special pair, P. Both mutants symmetrize amino acid residues so that portions of the M-sequence are replaced with L-sequence: sym1 symmetrizes all residues between M187 and M203, whereas (M)F195H is a single amino acid subset of the sym1 mutation. (M)F195H introduces a His residue in a position where it is likely to form a hydrogen bond to the acetyl group of the M-side bacteriochlorophyll of P. For both mutants compared with wild-type, (i) the redox potential is at least 100 meV greater, (ii) the P+QA- recombination rate is about twice as fast at room temperature, and (iii) the large electroabsorption feature for the QY band of P is shifted relative to the absorption spectrum. The comparison of the properties observed for the sym1 and (M)F195H reaction center mutants and the differences between these mutants and wild-type suggest that residue M195 is an important determinant of the properties of the special pair.

The cloning and mapping of ADR6, a gene required for sporulation and for expression of the alcohol dehydrogenase II isozyme from Saccharomyces cerevisiae.

The alcohol dehydrogenase II (ADH2) gene of the yeast, Saccharomyces cerevisiae, is not transcribed during growth on fermentable carbon sources such as glucose. Growth of yeast cells in a medium containing only nonfermentable carbon sources leads to a marked increase or derepression of ADH2 expression. The recessive mutation, adr6-1, leads to an inability to fully derepress ADH2 expression and to an inability to sporulate. The ADR6 gene product appears to act directly or indirectly on ADH2 sequences 3' to or including the presumptive TATAA box. The upstream activating sequence (UAS) located 5' to the TATAA box is not required for the Adr6- phenotype. Here, we describe the isolation of a recombinant plasmid containing the wild-type ADR6 gene. ADR6 codes for a 4.4-kb RNA which is present during growth both on glucose and on nonfermentable carbon sources. Disruption of the ADR6 transcription unit led to viable cells with decreased ADHII activity and an inability to sporulate. This indicates that both phenotypes result from mutations within a single gene and that the adr6-1 allele was representative of mutations at this locus. The ADR6 gene mapped to the left arm of chromosome XVI at a site 18 centimorgans from the centromere.

The identification and characterization of ADR6, a gene required for sporulation and for expression of the alcohol dehydrogenase II isozyme from Saccharomyces cerevisiae.

The alcohol dehydrogenase II isozyme (enzyme, ADHII; structural gene, ADH2) of the yeast, Saccharomyces cerevisiae, is under stringent carbon catabolite control. This cytoplasmic isozyme exhibits negligible activity during growth in media containing fermentable carbon sources such as glucose and is maximal during growth on nonfermentable carbon sources. A recessive mutation, adr6-1, and possibly two other alleles at this locus, were selected for their ability to decrease Ty-activated ADH2-6c expression. The adr6-1 mutation led to decreased ADHII activity in both ADH2-6c and ADH2+ strains, and to decreased levels of ADH2 mRNA. Ty transcription and the expression of two other carbon catabolite regulated enzymes, isocitrate lyase and malate dehydrogenase, were unaffected by the adr6-1 mutation. adr6-1/adr6-1 strains were defective for sporulation, indicating that adr6 mutations may have pleiotropic effects. The sporulation defect was not a consequence of decreased ADH activity. Since the ADH2-6c mutation is due to insertion of a 5.6-kb Ty element at the TATAA box, it appears that the ADR6+-dependent ADHII activity required ADH2 sequences 3' to or including the TATAA box. The ADH2 upstream activating sequence (UAS) was probably not required. The ADR6 locus was unlinked to the ADR1 gene which encodes another trans-acting element required for ADH2 expression.

Carbon source dependence of transposable element-associated gene activation in Saccharomyces cerevisiae.

Seven cis-dominant mutations leading to the overproduction of the glucose-repressible alcohol dehydrogenase isozyme ADHII (structural gene, ADH2) in Saccharomyces cerevisiae have previously been shown to be due to insertion of a transposable element, Ty, in the 5' regulatory region of the ADH2 gene. We showed that although mating-competent cells (a, alpha, a/a, or alpha/alpha cells) overproduced both ADHII enzyme and ADH2 mRNA, mating-incompetent cells (a/alpha or ste-cells) produced much less ADHII enzyme and ADH2 mRNA. This mating type effect on ADH2 expression was greatest in the presence of a normally derepressing carbon source, glycerol, and much less apparent in the presence of a repressing carbon source, glucose. In addition, Ty insertion led to an aberrant carbon source response in mating-incompetent cells--the normally glucose-repressible ADHII becomes glycerol repressible. The mating type effect and aberrant carbon source response in mating-incompetent cells was specific for Ty-associated mutations in the 5' flanking region of the ADH2 gene in that a non-Ty mutation in the same region did not show these effects. Finally, Ty1 RNA levels also showed a/alpha, suppression, which was apparent only during growth on a nonfermentable carbon source such as glycerol. This suggests that Ty-mediated gene expression is subject to regulation by both mating competence and carbon catabolites.