Slow Fluorescence Change Research Articles

Fluorescence lifetime imaging microscopy of Chlamydomonas reinhardtii: non‐photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transient

Fluorescence lifetime-resolved images of chlorophyll fluorescence were acquired at the maximum P-level and during the slower transient (up to 250 s, including P-S-M-T) in the green photosynthetic alga Chlamydomonas reinhardtii. At the P-level, wild type and the violaxanthin-accumulating mutant npq1 show similar fluorescence intensity and fluorescence lifetime-resolved images. The zeaxanthin-accumulating mutant npq2 displays reduced fluorescence intensity at the P-level (about 25-35% less) and corresponding lifetime-resolved frequency domain phase and modulation values compared to wild type/npq1. A two-component analysis of possible lifetime compositions shows that the reduction of the fluorescence intensity can be interpreted as an increase in the fraction of a short lifetime component. This supports the important photoprotection function of zeaxanthin in photosynthetic samples, and is consistent with the notion of a 'dimmer switch'. Similar, but quantitatively different, behaviour was observed in the intensity and fluorescence lifetime-resolved imaging measurements for cells that were treated with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, the efficient PSI electron acceptor methyl viologen and the protonophore nigericin and. Lower fluorescence intensities and lifetimes were observed for all npq2 mutant samples at the P-level and during the slow fluorescence transient, compared to wild type and the npq1 mutant. The fluorescence lifetime-resolved measurements during the slow fluorescence changes after the P level up to 250 s for the wild type and the two mutants, in the presence and absence of the above inhibitors, were analyzed with a graphical procedure (polar plots) to determine lifetime compositions. At higher illumination intensity, wild type and npq1 cells show a rise in fluorescence intensity and corresponding rise in the species concentration of the slow lifetime component after the initial decrease following the P level. This reversal is absent in the npq2 mutant, and for all samples in the presence of the inhibitors. Lifetime heterogeneities were observed in experiments averaged over multiple cells as well as within single cells, and these were followed over time. Cells in the resting state (induced by several hours of darkness), instead of the normal swimming state, show shortened lifetimes. The above results are discussed in terms of a superposition of effects on electron transfer and protonation rates, on the so-called 'State Transitions', and on non-photochemical quenching. Our data indicate two major populations of chlorophyll a molecules, defined by two 'lifetime pools' centred on slower and faster fluorescence lifetimes.

Use of Viscogens, dNTPαS, and Rhodium(III) as Probes in Stopped-Flow Experiments To Obtain New Evidence for the Mechanism of Catalysis by DNA Polymerase β,

The kinetic mechanism and the structural bases of the fidelity of DNA polymerases are still highly controversial. Here we report the use of three probes in the stopped-flow studies of Pol beta to obtain new, direct evidence for our previous interpretations: (a) Increasing the viscosity of the reaction buffer by sucrose or glycerol is expected to slow down the conformational change differentially, and it was shown to slow down the first (fast) fluorescence transition selectively. (b) Use of dNTPalphaS in place of dNTP is expected to slow down the chemical step preferentially, and it was shown to slow down the second (slow) fluorescence transition selectively. (c) The substitution-inert Rh(III)dNTP was used to show for the first time that the slow fluorescence change occurs after mixing of Pol beta.DNA.Rh(III)dNTP with Mg(II). These results, along with crystal structures, suggest that the subdomain-closing conformational change occurs before binding of the catalytic Mg(II) while the rate-limiting step occurs after binding of the catalytic Mg(II). These results provide new evidence to the mechanism we suggested previously, but do not support the results of three recent papers of computational studies. The results were further supported by a "sequential mixing" stopped-flow experiment that used no analogues, and thus ruled out the possibility that the discrepancy between experimental and computational results is due to the use of analogues. The methodologies can be used to examine other DNA polymerases to answer whether the properties of Pol beta are exceptional or general.

DNA Polymerase β. 5. Dissecting the Functional Roles of the Two Metal Ions with Cr(III)dTTP1

DNA polymerases catalyze the synthesis of DNA with a fidelity ranging from 1000 to 100 000.2 Although the kinetic properties of DNA polymerases have been investigated extensively,3-5 the physical origin of this high fidelity is still poorly understood. The crystal structures of DNA polymerase â (Pol â) from rat brain show the presence of two Mg2+ ions in the active site6a,b,7 (Figure 1). Functionally, it has been established that DNA polymerases require divalent metal ions for catalysis.8,9 However, no kinetic evidence that two divalent metal ions are required has been obtained for DNA polymerases, nor have the roles of the two metal ions been dissected functionally. We here report the use of Cr(III)dNTP coupled with stopped-flow fluorescence to show the requirement of two metal ions, and to dissect their functional roles in the catalysis by Pol â from rat brain (overexpressed in Escherichia coli).5 Pol â can serve as a good model for nucleotidyl transfer reactions because of its small size (39 kDa) and simplicity (without exonuclease or proof-reading activities). Using a synthetic DNA primer/template containing the 2-aminopurine nucleotide analogue (2-AP) (Figure 2A), we recently reported two phases of fluorescence changes in the stopped-flow fluorescence assay of nucleotide incorporation catalyzed by Pol â (Figure 2B).10 The fast phase was attributed to a conformational change step because its rate dependence on [dTTP] is hyperbolic. The rate of the slow phase corresponds to product formation. With a dideoxynucleotide-terminated primer, we have successfully dissected the slow fluorescence change and the chemical step, and attributed the slow phase to a conformational change that is the rate-limiting step in the reaction.10 Cr(III)‚nucleotide complexes have been extensively used as analogues of Mg(II)‚nucleotide complexes to solve enzymological problems because of their exchange-inert properties.11-13 When the Pol â‚DNA complex was mixed with â,γ-bidentate Cr(ΙΙΙ)dTTP14 in the absence of Mg2+, only the fast phase was observed (Figure 2C). However, the slow phase was restored upon addition of Mg2+ (Figure 2D). Under the latter condition the substrates were turned over, and the rate of the slow phase was comparable to the rate of product (21-mer) formation measured by the rapid chemical quench assay. In the absence of any metal ions (dTTP alone) neither fluorescence changes nor catalysis occurred. These results clearly indicate that the fast conformational change is induced by the binding of Cr(III)dTTP and the slow conformational change by the binding of Mg2+. On the basis of the crystal structure shown in Figure 1, Cr3+ and Mg2+ should occupy the metal ion sites A and B, respectively.6c The results described above, taken together with our pre-steadystate kinetic analyses reported previously, strongly support the mechanism we proposed for Pol â:10

Is there a rate-limiting step before GTP cleavage by H-ras p21?

A slow fluorescence change of the complex between ras p21 and the fluorescent GTP analogue 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-triphosphate (mGTP) has been postulated to be a signal arising from a step which is rate limiting and precedes the actual GTP hydrolysis reaction [Neal, S. E., Eccleston, J. F., & Webb, M. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565]. We have now shown that the rate of the fluorescence change is accelerated by GTPase-activating protein (GAP) in the same manner as that of the GTP cleavage reaction. In contrast, a faster fluorescence change of smaller amplitude seen in the complex between p21 and the uncleavable 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-O-(beta,gamma-imidotriphosphate) (mGppNHp) is not affected by GAP. The corresponding fluorescent derivative of guanosine 5'-O-(gamma-thiotriphosphate) (mGTP gamma S) shows a very slow fluorescence change after binding to p21, and this rate is also accelerated significantly by GAP. Hydrolysis of GTP gamma S is similarly slow, and it is accelerated by GAP in a similar manner to the fluorescence change. The results are interpreted to indicate that the fluorescence change occurs either at the hydrolysis step or on release of inorganic phosphate or thiophosphate but does not occur in a rate-limiting step preceding hydrolysis.

GTP hydrolysis mechanisms in ras p21 and in the ras-GAP complex studied by fluorescence measurements on tryptophan mutants

We have substituted leucine 56 or tyrosine 64 of p21 ras with a tryptophan. The intrinsic fluorescence of this tryptophan was used as an internal conformational probe for time-resolved biochemical studies of the ras protein. The slow intrinsic GTPase, GDP/GTP exchange induced by the SDC25 "exchange factor", and the fast GTP hydrolysis induced by GAP were studied. Tryptophan fluorescence of mutated ras is very sensitive to magnesium binding, GDP/GTP exchange, and GTP hydrolysis (changes in tyrosine fluorescence of wild-type ras are also observed but with a lower sensitivity). Nucleotide affinities, exchange kinetics, and intrinsic GTPase rates of the mutated ras could be measured by this method and were found to be close to those of wild-type ras. The SDC25 gene product enhances GDP/GTP exchange in both mutants. In both mutants, a slow fluorescence change follows the binding of GTP gamma S; its kinetics are close to those of the intrinsic GTPase, suggesting that a slow conformational change precedes the GTPase and is the rate-limiting step, as proposed by Neal et al. (1990) (Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565). GAP interacts with both mutant ras proteins and accelerates the GTPase of (L56W)ras but not that of (Y64W)ras, suggesting a role for tyrosine 64 in GAP-induced GTP hydrolysis. However, GAP does not accelerate the slow conformational change following GTP gamma S binding in either of the mutated ras proteins. This suggests that the fast GAP-induced catalysis of GTP hydrolysis that is observed with (L56W)ras bypasses the slow conformational change associated with the intrinsic GTPase and therefore might proceed by a different mechanism.

Protein fluorescence changes associated with ATP and adenosine 5'-[gamma-thio]triphosphate binding to skeletal muscle myosin subfragment 1 and actomyosin subfragment 1.

1. The fluorescence changes accompanying the binding of ATP and adenosine 5'-[gamma-thio]triphosphate (ATP gamma S) to myosin subfragment 1 (S1) and actomyosin subfragment 1 (actoS1) have been reinvestigated at 20 degrees C and 1 degree C, pH 7.0, 0.1 M-KCl. 2. Two successive fluorescence enhancements are observed following ATP binding to both S1 and actoS1. 3. The slow fluorescence change has the same rate with S1 and actoS1, and is due to the ATP cleavage step. 4. With actoS1 the fast fluorescence change occurs after dissociation, so a new intermediate, S1 ATP, is required on the actoS1 pathway. 5. The dissociation of actoS1 by ATP gamma S results in a fluorescence enhancement with the same apparent rate as dissociation, but indirect evidence suggests that this too occurs on a dissociated state.

Fluorescence induction in Avena sativa investigated by modulated light

The structurated slow fluorescence induction of six days old seedlings of Avena sativa L., cv. Seger I (Svalöf, Sweden) has been investigated in its responses to systematic changes in the exciting conditions. The intensity of the exciting light was periodically altered between a high and a low value, and after the third period the resulting fluorescence induction curves became stationary. These stationary responses may all be resolved into four characteristic fluorescence transients, whose forms were rather independent of the exciting conditions. The heights of these transients were examined as a function of the exciting wavelength and the high level intensity. From these data it is concluded that the third transient originates from cyclic electron transport and that the fourth transient monitors the degree of energy transfer from the light harvesting complex to photosystem II. These two last transients of the induction curve are equivalent to the structurated slow fluorescence changes in ordinary induction curves.

Chlorophyll a fluorescence of Gonyaulax polyedra grown on a light-dark cycle and after transfer to constant light.

Abstract— The chlorophyll a fluorescence properties of Gonyaulax polyedra cells before and after transfer from a lightdark cycle (LD) to constant dim light (LL) were investigated. The latter display a faster fluorescence transient from the level ‘I’ (intermediary peak) to ‘D’ (dip) to ‘P’ (peak) than the former (3 s as compared to 10 s), and a different pattern of decline in fluorescence from ‘I’ to ‘D’ and from ‘P’ to the steady state level with no clearly separable second wave of slow fluorescence change, referred to as ‘s' (quasi steady state)→‘M’ (maximum) →‘T’ (terminal steady state). The above differences are constant features of cells in LD and LL, and are not dependent on the time of day. They are interpreted as evidence for a greater ratio of photosystem II/photosystem I activity in cells in LL.After an initial photoadaptive response following transfer from LD to LL, the cell absorbance at room temperature and fluorescence emission spectra at 77 K for cells in LL and LD are comparable. The major emission peak is at 685–688 nm (from an antenna Chl a 680, perhaps Chl a‐c complex), but, unlike higher plants and other algae, the emission bands at 696–698 nm (from Chl aII complex, Chl a 685, close to reaction center II) and 710–720 nm (from Chl a1, complexes, Chl a 695, close to reaction center I) are very minor and could be observed only in the fluorescence emission difference spectra of LL minus LD cells and in the ratio spectra of DCMU‐treated to non‐treated cells.Comparison of emission spectra of cells in LL and LD suggested that, in LL, there is a slightly greater net excitation energy transfer from the light‐harvesting peridinin‐Chl a (Chl a 670) complex, fluorescing at 675 nm, to the other antenna chlorophyll a complex fluorescing at 685–688 nm, and from the Chl a., complex to the reaction center II. Comparison of excitation spectra of fluorescence of LL and LD cells, in the presence of DCMU, confirmed that cells in LL transfer energy more extensively from the peridinin‐Chl a complex to other Chl a complexes than do cells in LD.

Intermediate states of subfragment 1 and actosubfragment 1 ATPase: reevaluation of the mechanism.

The kinetics of the increase in protein fluorescence following the addition of ATP to subfragment-1 (SF-1) and acto-SF-1 have been reinvestigated. The concentration dependence of the rate obtained with SF-1 did not fit a hyperbola and at high ATP concentration, approximately 40% of the signal amplitude was lost due to a fast phase at the beginning of the transient (20 degrees C). At lower temperature (less than or equal to 10 degrees C) the fluorescence transient was biphasic, with a fast phase observed at high ATP concentration. These results indicate that there are two steps in the SF-1 pathway in which there is a change in protein fluorescence. Measurements of ATP binding and hydrolysis by chemical quench-flow methods indicate that the rate of ATP binding is correlated with the fast fluorescence step and hydrolysis is correlated with the slow fluorescence change. The SF-1 mechanism can thus be described as: (formula: see text) where M represents SF-1 and states of enhanced fluorescence are given by M (16%) and M (36% enhancement, relative to SF-1). Step 1 is a rapid equilibrium with K1 approximately 10(3) M-1. Tight binding of ATP occurs in step 2 and the loss of signal amplitude requires k2 greater than or approximately 1500--2000 s-1. The maximum observed fluorescence rate defines the rate of hydrolysis, k3 + k-3 = 125 s-1 (20 degrees C, 0.1 M KCl, pH 7.0). The steps in the mechanism correspond to the Bagshaw--Trentham scheme, with the important difference that the assignment of rate constant is altered. Formation of the acto-SF-1 complex gave a fluorescence enhancement of approximately 14% relative to SF-1. Dissociation of acto-SF-1 by ATP produced a 20--22% enhancement in fluorescence. There was no detectable fluorescence change during dissociation as evidenced by a lag in the fluorescence transient which corresponded to the kinetics of dissociation. The fluorescence change occurred at the same maximum rate as for SF-1 but there was no loss in signal amplitude at high ATP concentration. The kinetics of the fluorescence change corresponded to the rate of ATP hydrolysis, whereas tight ATP binding occurred at a much faster rate in approximate agreement with the rate of dissociation. Thus the fluorescence change in the acto-SF-1 pathway corresponds to step 3 in the SF-1 mechanism. The complete scheme can be described as follows: (formula: see text) where AM represents acto-SF-1. The tight binding step in the SF-1 pathway (k2) is sufficiently fast so that a similar step (k2') in the acto-SF-1 pathway could precede dissociation but the AM-ATP intermediate has not been detected. Following hydrolysis on the free SF-1, actin recombines with M.ADP.Pi or possibly with a second SF-1 product intermediate as proposed by Chock et al. (1976) and the fluorescence returns to the original AM level with product release.

Rapid light-induced changes of energy distribution between photosystems I and II

Chlorophyll fluorescence induction phenomena (Kautsky effect) have been very useful in the study of photosynthesis (for a review see [ 11. It has long been assumed that the fluorescence induction kinetics are independent of measuring wavelength. Recently it was discovered in Butler’s laboratory that the F690/F730 ratio undergoes changes during the slower part of the induction period (R. J. Strasser, personal communication), where other investigators [2-41 had already observed fluorescence changes, independent of photosystem II acceptor Q*. These slowfluorescence changes are believed to reflect ion-induced changes in energy distribution between the two photosystems (for reviews see [ 1,5,6] ). Any increase in the energy fraction distributed to System I should lead to an increase of System I fluorescence centered around 735 nm relative to System II fluorescence at 685 nm. In earlier work we concluded that not only slow fluorescence changes but also certain rapid transients are due to changes of energy ‘spill-over’ from pigment system II to pigment system I [7,8]. If this conclusion is correct, rapid changes in the ratio of System I and System II fluorescence are also expected. With a system developed to monitor fluorescence ratios automatically and having a time resolution under 1 ms, we did discover very fast changes. Our new data support earlier conclusions of rapid energy distribution changes and suggest a ‘switch’ mechanism, which operates far faster than ion-induced conformational changes of the thylakoid membrane could occur.

Light-induced changes in the fluorescence yield of chlorophyll a in Anacystis nidulans. I. Relationship of slow fluorescence changes with structural changes

1. 1. Both normal and 3-(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned cells of the blue-green alga Anacystis nidulans show an extensive slow rise in the chlorophyll a fluorescence yield upon illumination (Papageorgiou and Govindjee, 1968). It was observed here that uncouplers of phosphorylation notably 5-chloro-3-( p-chlorophenyl)-4′-chlorosalicylanilide and 5-chloro-3- tert-butyl-2′-chloro-4′-nitrosalicylanilide suppress this slow rise in the fluorescence yield both in normal and poisoned samples. In comparison to these salicylanilides, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone seems to be less effective in this respect. 2. 2. Emission spectra measured with the salicylanilides in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells show that the uncouplers prevent a shift in the excitation transfer in favor of pigment system II, i.e. the transformation to the so-called “State 1”. In other words, in the presence of the uncoupler, the pigment systems remain arrested in “State 2”. According to this view, the slow fluorescence rise represents a shift from State 2 to State 1. 3. 3. Furthermore, the uncouplers suppress as well the light-induced structural alterations (as measured by a change in absorbance at 540 nm) in both normal and poisoned Anacystis cells. Fixation of cells with aldehydes, that immobilizes the structural alterations, also abolishes slow fluorescence yield changes. 4. 4. It was observed that the time course of the light-induced macroscopic structural alteration was slower as compared to slow fluorescence yield changes. It, therefore, seems that the microscopic conformational organization, that precedes the macroscopic volume changes, induces the slow changes in fluorescence yield by shifting the mode of excitation energy transfer. 5. 5. In conclusion, we suggest that an energy-dependent specific alteration of the organization between the two photosystems controls and regulates the mode of excitation energy transfer between the two photosystems, the efficiency of such a transfer in Anacystis is approximately 10%.

On the interpretation of energy linked 1-anilino-8-naphthalene sulfonic acid fluorescence changes in mitochondrial fragments

The study of the energy transfer system of mitochondrial membranes has recently taken advantage of the use of fluorescent probes [l-4] to monitor structural changes associated with energy conservation [S-9]. A fluorescence increase of 1 -anilinonaphthalene8-sulfonic acid (ANS) occurs when mitochondrial fragments are “energized” either from respiratory substrates, ATP or by the use of a cationic gradient [lo]. Such a fluorescence enhancement has been attributed to an increase in ANS binding as a consequence of an increased affinity of the membrane for the probe. In fact uncoupler and inhibitory sensitive binding changes can be measured by different techniques [ 7, lo] during cycles of “energization” and “de-energization” of the mitochondrial membrane. An alternative explanation of the fluorescence changes of ANS, namely an increased quantum yield [ 1 l] of bound probe during energy conservation in mitochondria has been proposed (cf. however [ 121). Those ANS molecules having a higher quantum yield would decay to a low fluorescent state upon uncoupling, in a rapid first order reaction preceding in time other molecules slowly leading out of the membrane [ 131. (Note however that the conclusion that the uncoupler induced fluorescence decay was the sum of two consecutive reactions was based on its analysis in terms of two paraZZeZ reactions with different rate constants [ 111). More recently, a mechanism was proposed [ 141 in which fast and slow fluorescence changes were considered the consequence of parallel first order reactions, in contrast with previous conclusions [ 131. More-