Effect of photosystem II reaction center closure on nanosecond fluorescence relaxation kinetics

The fluorescence decay of chlorophyll in spinach thylakoids was measured as a function of the degree of closure of Photosystem II reaction centers, which was set for the flowed sample by varying either the preillumination by actinic light or the exposure of the sample to the exciting pulsed laser light. Three exponential kinetic components originating in Photosystem II were fitted to the decays; a fourth component arising from Photosystem I was determined to be negligible at the emission wavelength of 685 nm at which the fluorescence decays were measured. Both the lifetimes and the amplitudes of the components vary with reaction center closure. A fast (170–330 ps) component reflects the trapping kinetics of open Photosystem II reaction centers capable of reducing the plastoquinone pool; its amplitude decreases gradually with trap closure, which is incompatible with the concept of photosynthetic unit connectivity where excitation energy which encounters a closed trap can find a different, possibly open one. For a connected system, the amplitude of the fast fluorescence component is expected to remain constant. The slow component (1.7–3.0 ns) is virtually absent when the reaction centers are open, and its growth is attributable to the appearance of closed centers. The middle component (0.4–1.7 ns) with approximately constant amplitude may originate from centers that are not functionally linked to the plastoquinone pool. To explain the continuous increase in the lifetimes of all three components upon reaction center closure, we propose that the transmembrane electric field generated by photosynthetic turnover modulates the trapping kinetics in Photosystem II and thereby affects the excited state lifetime in the antenna in the trap-limited case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PQ plastoquinone - PSI and PSII Photosystem I and II - Q_A and Q_B primary and secondary quinone acceptor of PSII     

The slow folding reaction of barstar: the core tryptophan region attains tight packing before substantial secondary and tertiary structure formation and final compaction of the polypeptide chain

The slow folding of a single tryptophan-containing mutant of barstar has been studied in the presence of 2 M urea at 10 degrees C, using steady state and time-resolved fluorescence methods and far and near-UV CD measurements. The protein folds in two major phases: a fast phase, which is lost in the dead time of measurement during which the polypeptide collapses to a compact form, is followed by a slow observable phase. During the fast phase, the rotational correlation time of Trp53 increases from 2.2 ns to 7.2 ns, and its mean fluorescence lifetime increases from 2.3 ns to 3.4 ns. The fractional changes in steady-state fluorescence, far-UV CD, and near-UV CD signals, which are associated with the fast phase are, respectively, 36 %, 46 %, and 16 %. The product of the fast phase can bind the hydrophobic dye ANS. These observations together suggest that the folding intermediate accumulated at the end of the fast phase has: (a) about 20 % of the native-state secondary structure, (b) marginally formed or disordered tertiary structure, (c) a water-intruded and mobile protein interior; and (d) solvent-accessible patches of hydrophobic groups. Measurements of the anisotropy decay of Trp53 suggest that it undergoes two types of rotational motion in the intermediate: (i) fast (tau(r) approximately 1 ns) local motion of its indole side-chain, and (ii) a slower (tau(r) approximately 7.2 ns) motion corresponding to global tumbling of the entire protein molecule. The ability of the Trp53 side-chain to undergo fast local motion in the intermediate, but not in the fully folded protein where it is completely buried in the hydrophobic core, suggests that the core of the intermediate is still poorly packed. The global tumbling time of the fully folded protein is faster at 5.6 ns, suggesting that the volume of the intermediate is 25 % more than that of the fully folded protein. The rate of folding of this intermediate to the native state, measured by steady-state fluorescence, far-UV CD, and near-UV CD, is 0.07(+/-0.01) min(-1) This rate compares to a rate of folding of 0.03(+/-0.005) min(-1), determined by double-jump experiments which monitor directly formation of native protein; and to a rate of folding of 0.05 min(-1), when determined from time-resolved anisotropy measurements of the long rotational correlation time, which relaxes from an initial value of 7.2 ns to a final value of 5. 6 ns as the protein folds. On the other hand, the amplitude of the short correlation time decreases rapidly with a rate of 0.24(+/-0.06) min(-1). These results suggest that tight packing of residues in the hydrophobic core occurs relatively early during the observable slow folding reaction, before substantial secondary and tertiary structure formation and before final compaction of the protein.     

Electron transfer in cyanobacterial photosystem I: II. Determination of forward electron transfer rates of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.     

Folding and assembly of dimeric human glutathione transferase A1-1

Glutathione transferases function as detoxification enzymes and ligand-binding proteins for many hydrophobic endogenous and xenobiotic compounds. The molecular mechanism of folding of urea-denatured homodimeric human glutathione transferase A1-1 (hGSTA1-1) was investigated. The kinetics of change were investigated using far-UV CD, Trp20 fluorescence, fluorescence-detected ANS binding, acrylamide quenching of Trp20 fluorescence, and catalytic reactivation. The very early stages of refolding (millisecond time range) involve the formation of structured monomers with native-like secondary structure and exposed hydrophobic surfaces that have a high binding capacity for the amphipathic dye ANS. Dimerization of the monomeric intermediates was detected using Trp fluorescence and occurs as fast and intermediate events. The intermediate event was distinguished from the fast event because it is limited by a preceding slow trans-to-cis isomerization reaction (optically silent in this study). At high concentrations of hFKBP, dimerization is not limited by the isomerization reaction, and only the fast event was detected. The fast (tau = 200 ms) and intermediate (tau = 2.5 s) events show similar urea-, temperature-, and ionic strength-dependent properties. The dimeric intermediate has a partially functional active site ( approximately 20%). Final reorganization to form the native tertiary and quaternary structures occurs during a slow, unimolecular, urea- and ionic strength-independent event. During this slow event (tau = 250 s), structural rearrangements at the domain interface occur at/near Trp20 and result in burial of Trp20. The slow event results in the regain of the fully functional dimer. The role of the C-terminus helix 9 (residues 210-221) as a structural determinant for this final event is proposed.     

Gating current kinetics in Myxicola giant axons. Order of the back transition rate constants.

Gating current, Ig, was recorded in Myxicola axons with series resistance compensation and higher time resolution than in previous studies. Ig at ON decays as two exponentials with time constants, tau ON-F and tau ON-S, very similar to squid values. No indication of an additional very fast relaxation was detected, but could be still unresolved. Ig at OFF also displays two exponentials, neither reflecting recovery from charge immobilization. Deactivation of the two I(ON) components may proceed with well-separated exponentials at -100 mV. INa tail currents at OFF also display two exponentials plus a third very slow relaxation of 5-9% of the total tail current. The very slow component is probably deactivation of a very small subpopulation of TTX sensitive channels. A -100 mV, means for INa tail component time constants (four axons) are 76 microseconds (range: 53-89 microseconds) and 344 microseconds (range: 312-387 microseconds), and for IOFF (six axons) 62 microseconds (range: 34-87 microseconds) and 291 microseconds (range: 204-456 microseconds) in reasonable agreement. INa ON activation time constant, tau A, is clearly slower than tau ON-F at all potentials. Except for the interval -30 to -15 mV, tau A is clearly faster than tau ON-S, and has a different dependency on potential. tau ON-S is several fold smaller than tau h. Computations with a closed2----closed1----open activation model indicated Na tail currents are consistent with a closed1----open rate constant greater than the closed2----closed1.     

The emission yields of delayed and prompt fluorescence from chloroplasts.

1. The decay of delayed fluorescence from chloroplasts blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and uncoupled with gramicidin has been measured in the time range 0.75--45 ms by use of a laser phosphoroscope. 2. The decays have been analysed as the sum of three first-order components of approximate half-lives 0.2, 2.5 and 300 ms by a computer-assisted least-squares fit procedure. 3. The prompt fluorescence yield of the chloroplasts was manipulated by changing the cation concentration of the chloroplast-suspending medium. 4. Analysis of the concentration dependence of the components of the delayed fluorescence decay and of the prompt fluorescence inductions indicates that the emission yield of the intermediate (tau approximately 2.5 ms) component of the decay is equal to the fluorescence yield of a Photosystem II photosynthetic unit with an open trap, and that for the slow (tau approximately 300 ms) component the emission yield is equal to the total Photosystem II prompt fluorescence yield. 5. It is concluded that the delayed fluorescence yield in the time range studied is a complex function of time, which may be due to there being different mechanisms leading to delayed fluorescence production at short and long times after cessation of illumination.     

Fluorescence anisotropy studies of molecularly imprinted polymers.

A molecularly imprinted polymer (MIP) is a biomimetic material that can be used as a biochemical sensing element. We studied the steady-state and time-resolved fluorescence and fluorescence anisotropy of anthracene-imprinted polyurethane. We compared MIPs with imprinted analytes present, MIPs with the imprinted analytes extracted, MIPs with rebound analytes, non-imprinted control polymers (non-MIPs) and non-MIPs bound with analytes to understand MIP's binding behaviour. MIPs and non-MIPs had similar steady-state fluorescence anisotropy in the range 0.11-0.24. Anthracene rebound in MIPs and non-MIPs had a fluorescence lifetime of tau = 0.64 ns and a rotational correlation time of phi(F) = 1.2-1.5 ns, both of which were shorter than that of MIPs with imprinted analytes present (tau = 2.03 ns and phi(F) = 2.7 ns). The steady-state anisotropy of polymer solutions increased exponentially with polymerization time and might be used to characterize the polymerization extent in situ.     

Effect of ajmaline on transient outward current in rat ventricular myocytes

The mechanism of ajmaline-induced inhibition of the transient outward current (I(to)) has been investigated in right ventricular myocytes of rat using the whole cell patch clamp technique. Ajmaline decreased the amplitude and the time integral of I(to) in a concentration-dependent, but frequency- and use-independent manner. In contrast to the single exponential time course of I(to)-inactivation in control conditions (tau(i) = 37.1 +/- 2.7 ms), the apparent inactivation was fitted by a sum of two exponentials under the effect of ajmaline with concentration-dependent fast and slow components (tau(f) = 11.7 +/- 0.8 ms, tau(s) = 57.6 +/- 2.7 ms at 10 micromol/l) suggesting block development primarily in the open channel state. An improved expression enabling to calculate the association and dissociation rate constants from the concentration dependence of tau(f) and tau(s) was derived and resulted in k(on) = 4.57 x 10(6) +/- 0.32 x 10(6) mol(-1).l.s(-1) and k(off) = 20.12 +/- 5.99 s(-1). The value of K(d) = 4.4 micromol/l calculated as k(off) / k(on) was considerably lower than IC(50) = 25.9 +/- 2.9 micromol/l evaluated from the concentration dependence of the integrals of I(to). Simulations on a simple model combining Hodgkin-Huxley type gating kinetics and drug-channel interaction entirely in open channel state agreed well with the experimental data including the difference between the K(d) and IC(50). According to the model, the fraction of blocked channels increases upon depolarization and declines if depolarization is prolonged. The repolarizing step induces recovery from block with time constant of 52 ms. We conclude that in the rat right ventricular myocytes, ajmaline is an open channel blocker with fast recovery from the block at resting voltage.     

Quenching of chlorophyll fluorescence by triplets in solubilized light-harvesting complex II (LHCII)

The quenching of chlorophyll fluorescence by triplets in solubilized trimeric light harvesting complexes was analyzed by comparative pump-probe experiments that monitor with weak 2-ns probe pulses the fluorescence yield and changes of optical density, DeltaOD, induced by 2-ns pump pulses. By using a special array for the measurement of the probe fluorescence (Sch?del R., F. Hillman, T. Schr?tter, K.-D. Irrgang, J. Voight, and G. Biophys. J. 71:3370-3380) the emission caused by the pump pulses could be drastically reduced so that even at highest pump pulse intensities, IP, no significant interference with the signal due to the probe pulse was observed. The data obtained reveal: a) at a fixed time delay of 50 ns between pump and probe pulse the fluorescence yield of the latter drastically decreased with increasing IP, b) the recovery of the fluorescence yield in the microseconds time domain exhibits kinetics which are dependent on IP, c) DeltaOD at 507 nm induced by the pump pulse and monitored by the probe pulse with a delay of 50 ns (reflecting carotenoid triplets) increases with IP without reaching a saturation level at highest IP values, d) an analogous feature is observed for the bleaching at 675 nm but it becomes significant only at very high IP values, e) the relaxation of DeltaOD at 507 nm occurs via a monophasic kinetics at all IP values whereas DeltaOD at 675 nm measured under the same conditions is characterized by a biphasic kinetics with tau values of about 1 microseconds and 7-9 microseconds. The latter corresponds with the monoexponential decay kinetics of DeltaOD at 507 nm. Based on a Stern-Volmer plot, the time-dependent fluorescence quenching is compared with the relaxation kinetics of triplets. It is shown that the fluorescence data can be consistently described by a quenching due to triplets.     

Tail currents in the myelinated axon of Xenopus laevis suggest a two-open-state Na channel.

Na tail currents in the myelinated axon of Xenopus laevis were measured at -70 mV after steps to -10 mV. The tail currents were biexponential, comprising a fast and a slow component. The time constant of the slow tail component, analyzed in the time window 0.35-0.50 ms, was independent of step duration, and had a value of 0.23 ms. The amplitude, extrapolated back to time 0, varied, however, with step duration. It reached a peak after 0.7 ms and inactivated relatively slowly (at 2.1 ms the absolute value was reduced by approximately 30%). The amplitude of the fast component, estimated by subtracting the amplitude of the slow component from the calculated total tail current amplitude, reached a peak (three times larger than that of the slow component) after 0.5 ms and inactivated relatively fast (at 2.1 ms it was reduced by approximately 65%). The results were explained by a novel Na channel model, comprising two open states bifurcating from a common closed state and with separate inactivating pathways. A voltage-regulated use of the two pathways explains a number of findings reported in the literature.     

Coupled kinetic traps in cytochrome c folding: His-heme misligation and proline isomerization

The effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH<5, the observed refolding at final pH 6 is dominated by a fast phase (tau(2f)=20 ms, alpha(2f)=90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (alpha(2f) approximately 20 %) but the same rate (tau(2f)=20 ms), and in two slower phases (tau(m)=6-8 seconds, alpha(m) approximately 45 %; and tau(1b)=16-20 seconds, alpha(1b) approximately 35 %). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand.     

Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?

Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a approximately 12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield ( approximately 50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by approximately 20-fold and that for closed PS I (with P700 oxidized) is increased by approximately 10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 +/- 1 ps for open PS I and 37 +/- 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing approximately 50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced.     

Kinetics of oxygen consumption after a single flash of light in photoreceptors of the drone (Apis mellifera)

The time course of the rate of oxygen consumption (QO2) after a single flash of light has been measured in 300-micrometers slices of drone retina at 22 degrees C. To measure delta QO2(t), the change in QO2 from its level in darkness, the transients of the partial pressure of O2 (PO2) were recorded with O2 microelectrodes simultaneously in two sites in the slice and delta QO2 was calculated by a computer using Fourier transforms. After a 40-ms flash of intense light, delta QO2, reached a peak of 40 microliters O2/g.min and then declined exponentially to the baseline with a time constant tau 1 = 4.96 +/- 0.49 s (SD, n = 10). The rising phase was characterized by a time constant tau 2 = 1.90 +/- 0.35 s (SD, n = 10). The peak amplitude of delta QO2 increased linearly with the log of the light intensity. Replacement of Na+ by choline, known to decrease greatly the light-induced transmembrane current, caused a 63% decrease of delta QO2. With these changes, however, the kinetics of delta QO2 (t) were unchanged. This suggest that the recovery phase is rate-limited by a single reaction with apparent first-order kinetics. Evidence is provided that suggests that this reaction may be the working of the sodium pump. Exposure of the retina to high concentrations of ouabain or strophanthidin (inhibitors of the sodium pump) reduced the peak amplitude of delta QO2 by approximately 80% and increased tau 1. The increase of tau 1 was an exponential function of the time of exposure to the cardioactive steroids. Hence, it seems likely that the greatest part of delta QO2 is used for the working of the pump, whose activity is the mechanism underlying the rate constant of the descending limb of delta QO2 (t).     

Fluorescence and oxygen evolution from Chlorella pyrenoidosa

The process of photosynthetic energy conversion in Chlorella pyrenoidosa was investigated by simultaneous measurement of transient and steady-state rates of O₂ evolution and fluorescence.

1. 1. Alternation or superimposition of light 1 and light 2 illumination induces both fast and slow changes in fluorescence and rate of O₂ evolution. The fast changes are ascribed to changes in conditions of the reaction centers in the context of the ¹ model and the kinetic analysis of ². The slow changes are interpreted as adaptations to the intensity and wavelength of illumination. The adaptive mechanism is described in terms of slow variation in fraction () of total absorbed quanta delivered to System 2. At low intensities, the calculated value of for cells adapted to light 2 illumination (light 2 state) is approx. 0.9 of for cells adapted to light 1 illumination (light 1 state).

2. 2. An increase in fluorescence yield was found to accompany the decrease in O₂ yield at the onset of light saturation with either light 1 or light 2 excitation. Variation in is proposed to account for the differences between the maximum fluorescence yield observed in steady-state conditions and the 1.5 times higher maximum yield observed in transient conditions or in cells inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. Variation in can also explain the observation of a higher rate of fluorescence emission with light 1 excitation than with light 2 excitation for a given steady-state rate of O₂ evolution.

3. 3. A model for energy conversion by System 2 is proposed to account for our observations. The model proposes competitive dissipation of absorbed energy by photochemical trapping at reaction centers and by fluorescence and radiationless de-excitation from both the pigment bed and reaction centers of System 2.

Induction patterns of delayed luminescence from isolated chloroplasts. I. Response of delayed luminescence to changes in the prompt fluorescence yield

1. Using a phosphoroscope, delayed luminescence and prompt chlorophyll fluorescence from isolated chloroplasts have been compared during the induction period.2. Two distinct decay components of delayed luminescence were measured a “fast” component (from ≈1 ms to ≈6 ms) and a “slow” component (at ≈6 ms).3. The fast luminescence component often did not correlate with the fluorescence changes while the slow component significantly changed its intensity during the induction period in a manner which could usually be linearly correlated with variable portion of the fluorescence yield change.4. This correlation was evident after preillumination with far-red light or after allowing a considerable time for dark relaxation.5. The close relationship between the slow luminescence component and variable fluorescence yield was observed with a large range of light intensities and also in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea which considerably changes the fluorescence induction kinetics.6. Valinomycin and other antibiotics reduced the amplitude of the 6 ms (slow) luminescence without affecting its relation with the fluorescence induction suggesting possibly that a constant electrical gradient exist in the dark or formed very rapidly in the light, which effects the emission intensity.7. Changes in salt levels of suspending media equally affected the amplitude of both delayed luminescence and variable fluorescence under conditions when the reduction of Q is maximal and constant.8. The results are discussed in terms of several models. It is concluded that the model of independent Photosystem II units together with photosynthetic back reaction concept is incompatible with the data. Other alternative models (the “lake” model and photosynthetic back reaction; recombination of charges in the antenna chlorophyll; the “W” hypothesis) were in closer agreement with the results.     

Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1

Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta(3)-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i. e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low ( approximately 1 M) GdnHCl concentrations.     

Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of Photosystem I fluorescence

The time-resolved fluorescence emission and excitation spectra of Chlorella vulgaris cells have been measured by single-photon timing with picosecond resolution. In a three-exponential analysis the time-resolved excitation spectra recorded at 685 and 706 nm emission wavelength with closed PS II reaction centers show large variations of the preexponential factors of the different decay components as a function of wavelength. At λ_em = 685 nm the major contribution to the fluorescence decay originates from two components with life-times of 2.1–2.4 and 1.2–1.3 ns. A short-lived component with life-times of 0.1–0.16 ns of relatively small amplitude is also found. When the emission is detected at 706 nm, the short-lived component with a life-time of less than 0.1 ns predominates. Time-resolved emission spectra using λ_exc = 630 or λ_exc = 652 nm show a spectral peak of the two longer-lived components at about 680–685 nm, whereas the fast component is red-shifted as compared to the others and shows a maximum at about 690 nm. The emission spectrum observed upon excitation at 696 nm with closed PS II reaction centers shows a large increase in the amplitude of the fast component with a lifetime of 80–100 ps as compared to that at 630 nm excitation. At almost open Photosystem II (PS II) reaction centers (F₀), the life-time of the fast component decreased from 150–160 ps at 682 nm to less than 100 ps at 720 nm emission wavelength. We conclude that at least two pigment pools contribute to the fast component. One is attributed to PS II and the other to Photosystem I (PS I). They have life-times of approx. 180 ps and 80 ps, respectively. The 80 ps (PS I) contribution has a spectral maximum slightly below 700 nm, whereas the 180 ps (PS II) spectrum peaks at 680–685 nm. The spectra of the middle decay component τ_m and its sensitivity to inhibitors of PS II suggest that this component is not preferentially related to LHC II but arises mainly from Chl a pigments probably associated with a second type of PS II centers. The amplitudes of the fast (180 ps, PS II) component and the long-lived decay show an opposite dependence on the state of the PS II centers and confirm our earlier conclusion that the contribution of PS II to the fast component probably disappears at the F_max state (Haehnel W., Holzwarth, A.R. and Wendler, J. (1983) Photochem. Photobiol. 34, 435–443). Our data are discussed in terms of α,β-heterogeneity in PS II centers.     

Time-resolved chlorophyll fluorescence studies on photosynthetic mutants of Chlamydomonas reinhardtii: origin of the kinetic decay components

The room temperature chlorophyll fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardtii have been measured as a function of Photosystem 2 (PS2) trap closure, DNB-induced quenching at F_M, and time-resolved emission spectra. The overall decays have been analyzed in terms of three or four kinetic components where necessary. A comparison of the characteristics of the decay components exhibited by the mutants with the wild-type has been carried out to elucidate the precise origins of the different emissions in relation to the observed pigment-protein complexes. It is shown that a) charge recombination in PS2 is not necessary for the presence of long-lived decay components, b) there are two rapid PS1-associated emissions (=30 and 150–200 ps), c) a slow PS1 decay is observed (=1.73 ns) in the absence of PS1 reaction centres, d) the two variable components (=0.25–1.2 and 0.5–2.2 ns) observed in the wild-type arise from LHC2 and e) a rapid (=50–250 ps) decay is associated with the PS2 core antenna (CP3 and CP4). These results show that the intact thylakoid membrane system is too complex to distinguish all of the individual kinetic components.Abbreviations Aexp preexponential factor (Amplitude) - chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNB m, dinitrobenzene - F_M maximum chl fluorescence level - F₀ initial chl fluorescence level - F_v variable chl fluorescence (F_M–F₀) - LHC light harvesting chl a/b protein complex - PS photosystem - Q_A primary stable electron acceptor of PS2     

The importance of life-time changes in fluorescence depolarization

The rotational relaxation time, rho, calculated from measurements of fluorescence depolarization, is clearly dependent on the assumed mean life-time, tau, of the excited state. However, variations in tau with experimental conditions (temperature and solvent composition) occur and the effect of such alterations in tau is demonstrated. In particular it should be noted that, unless life-time changes can be excluded, the occurrence of linear plots of reciprocal degree of polarization against the temperature/viscosity ratio does not necessarily indicate the absence of intramolecular freedoms. An attempt to correct for such life-time changes by measurement of the fluorescence intensity is made for the bovine serum albumin-1-dimethyl-aminonaphthalene-5-sulphonyl chloride system. The value of rho/3tau thus obtained for this system at 20 degrees is approx. 4.7, as against approx. 3.4 obtained by several workers in the absence of life-time corrections.