Determination of the excited-state lifetimes of the tryptophan residues in barnase, via multifrequency phase fluorometry of tryptophan mutants.

A multifrequency phase fluorometric study is described for wild-type barnase and engineered mutant proteins in which tryptophan residues have been replaced by less fluorescent residues which do not interfere with the determination of the tryptophan emission spectra and lifetimes. The lifetimes of the three tryptophans in the wild-type protein have been resolved. Trp-35 has a single fluorescence lifetime, which varies in the different proteins between 4.3 and 4.8 ns and is pH-independent between pH 5.8 and 8.9. Trp-71 and Trp-94 behave as an energy-transfer couple with both forward and reverse energy transfer. The couple shows two fluorescence lifetimes: 2.42 (+/-0.2) and 0.74 (+/-0.1) ns at pH 8.9, and 0.89 (+/-0.05) and 0.65 (+/-0.05) ns at pH 5.8. In the mutant Trp-94----Phe the lifetime of Trp-71 is 4.73 (+/-0.008) ns at high pH and 4.70 (+/-0.004) ns at low pH. In the mutant Trp-71----Tyr, the lifetime of Trp-94 is 1.57 (+/-0.01) ns at high pH and 0.82 (+/-0.025) ns at low pH. From these lifetimes, one-way energy-transfer efficiencies can be calculated according to Porter [Porter, G.B. (1972) Theor. Chim. Acta 24, 265-270]. At pH 8.9, a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was 86% for forward and 4% for reverse transfer (all +/-2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of F?rster [F?rster, T. (1948) Ann. Phys. (Leipzig) 2, 55-75].(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved fluorescence studies of tomaymycin bonding to synthetic DNAs.

Tomaymycin reacts covalently with guanine in the DNA minor groove, exhibiting considerable specificity for the flanking bases. The sequence dependence of tomaymycin bonding to DNA was investigated in synthetic DNA oligomers and polymers. The maximum extent of bonding to DNA is greater for homopurine and natural DNA sequences than for alternating purine-pyrimidine sequences. Saturation of DNA with tomaymycin has little effect on the melting temperature in the absence of unbound drug. Fluorescence lifetimes were measured for DNA adducts at seven of the ten unique trinucleotide bonding sites. Most of the adducts had two fluorescence lifetimes, representing two of the four possible binding modes. The lifetimes cluster around 2-3 ns and 5-7 ns; the longer lifetime is the major component for most bonding sites. The two lifetime classes were assigned to R and S diastereomeric adducts by comparison with previous NMR results for oligomer adducts. The lifetime difference between binding modes is interpreted in terms of an anomeric effect on the excited-state proton transfer reaction that quenches tomaymycin fluorescence. Bonding kinetics of polymer adducts were monitored by fluorescence lifetime measurements. Rates of adduct formation vary by two orders of magnitude with poly(dA-dG).poly(dC-dT), reacting the fastest at 4 x 10(-2) M-1 s-1. The sequence specificity of tomaymycin is discussed in light of these findings and other reports in the literature.     

Conformational effects on tryptophan fluorescence in cyclic hexapeptides

The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.     

Conformation and dynamics of bovine brain S-100a protein determined by fluorescence spectroscopy.

We have used time-resolved laser fluorescence spectroscopy to investigate the intensity and anisotropy decays of the single tryptophan residue in bovine brain S-100a (alpha beta) protein. The steady-state and acrylamide quenching results indicated that the Trp 90 of the alpha-subunit was partially buried in a relatively nonpolar environment at pH 7.5. Both Ca2+ and pH 8.5 slightly enhanced the exposure of the residue to the solvent, but the residue remained partially buried in the calcium complex at both pH values. The best representation of the intensity decays was a linear combination of three exponential terms, regardless of solvent condition and temperature. The three lifetimes (tau i) were in the range of 0.4-5 ns and insensitive to emission wavelength, but their fractional amplitudes (alpha i) shifted in favor of the shortest component (alpha 1) when the decays were measured at the blue end of the emission spectrum. These results suggest that an excited-state interaction between the indole ring and the side chain of an adjacent residue may be responsible for the observed shortest lifetime. In the presence of Ca2+, the three lifetimes remained relatively unaltered, but the values of alpha 1 decreased by a factor of 2.3 at pH 7.2 and a factor of 1.8 at pH 8.2. This Ca(2+)-induced decrease may be attributed to disruption of the putative excited-state interaction resulting from reorientations of the alpha-helical segments flanking a Ca(2+)-binding loop (residues 62-73). At both pH 7.2 and 8.4, the anisotropy decays of the apoprotein followed a biexponential decay law.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell

Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image. The nanosecond excited-state lifetime is independent of probe concentration or light path length but dependent upon excited-state reactions such as fluorescence resonance energy transfer (FRET). These properties of fluorescence lifetimes allow exploration of the molecular environment of labelled macromolecules in the interior of cells. Imaging of fluorescence lifetimes enables biochemical reactions to be followed at each microscopically resolvable location within the cell.     

Picosecond fluorescence of cryptomonad biliproteins. Effects of excitation intensity and the fluorescence decay times of phycocyanin 612, phycocyanin 645, and phycoerythrin 545.

The fluorescence of purified biliproteins (phycocyanin 645, phycocyanin 612, and phycoerythrin 545) from three cryptomonads, Chroomonas species, Hemiselmis virescens, and Rhodomonas lens, and C-phycocyanin from Anacystis nidulans has been time resolved in the picosecond region with a streak camera system having less than or equal to 2-ps jitter. The fluorescence lifetimes of phycocyanins from Chroomonas species and Hemiselmis virescens are 1.5 +/- 0.2 ns and 2.3 +/- 0.2 ns, respectively, regardless of the fluence of the 30 ps, 532-nm excitation pulse. (Fluence [or photons/cm2] = f intensity [photons/cm2s]dt.). In contrast, that of C-phycocyanin is 2.3 +/- 0.2 ns when the excitation fluence is 8.2 X 10(11) photons/cm2 and decreases to a decay approximated by an exponential decay time of 0.65 +/- 0.1 ns at 7.2 X 10(16) photons/cm2. The cryptomonad phycoerythrin fluorescence decay lifetime is also dependent on intensity, having a decay time of 1.5 +/- 0.1 ns at low fluences and becoming clearly biphasic at higher fluences (greater than 10(15) photons/cm2). We interpret the shortening of decay times for C-phycocyanin and phycoerythrin 545 in terms of exciton annihilation, and have discussed the applicability of exciton annihilation theories to the high fluence effects.     

Tryptophan fluorescence lifetimes in lysozyme.

Tryptophan fluorescence lifetimes at pH 2 and pH 8 have been obtained for lysozyme and for lysozyme derivatives in which tryptophan-62 or tryptophan-108 or both are nonfluorescent. The lifetimes range from about 0.5 ns to 2.8 ns for the various emitting tryptophans. The tryptophan lifetimes appear to increase with exposure of tryptophan to solvent, but intramolecular contacts, probably with cystine residues, can considerably shorten the lifetime. Intertryptophanyl interactions can also affect fluorescence lifetimes. The trytophan-108 lifetime in lysozyme is shorter than in the derivative in which tryptophan-62 is oxidized; this is ascribed to energy transfer from tryptophan-108 to tryptophan-62. From the lifetime results the relative intensities emitted by specific tryptophans can be estimated, and these values also support the existence of intertryptophanyl energy transfer. The emission intensity from tryptophan-62 is greater in the presence of tryptophan-108, and the emission intensity of tryptophan-108 appears to be greater in the absence of tryptophan-62. Conformational effects accompanying chemical modification of tryptophan cannot be completely ruled out, however. The tryptophan-62 lifetime at pH 8 in lysozyme is shorter than in the derivatives, which might indicate a subtle conformational effect. Studies with tri-(N-acetyl-glucosamine)-protein complexes indicate that both the tryptophan lifetimes and the number of emitting tryptophans may be changing upon complexation. The results illustrate the usefulness and the limitations of lifetime measurements in understanding protein fluorescence.     

Phenylalanine-to-tyrosine singlet energy transfer in the archaebacterial histone-like protein HTa

The Archaebacterium Thermoplasma acidophilum has a histone-like protein (HTa) abundantly associated with its deoxyribonucleic acid. Each native tetrameric complex of HTa contains 20 phenylalanine residues, 4 tyrosine residues, and no tryptophan. When the protein was excited by radiation at 252 nm, which is a wavelength absorbed predominantly by phenylalanine, the fluorescent emission was mostly from tyrosine. According to the excitation spectrum for this tyrosine fluorescence, the cause was energy transfer from phenylalanine, which occurred with about 50% efficiency. When the tyrosine residues were removed enzymatically, the excited-state lifetime of the phenylalanine residues nearly doubled. Because of energy transfer, the tyrosine emission had two apparent fluorescence decay lifetimes; one lifetime (3.9 ns) was that of tyrosine while the second (12.1 ns) corresponded to the excited state of phenylalanine.     

Conformational transitions in the calcium adenosinetriphosphatase studied by time-resolved fluorescence resonance energy transfer

We have used time-resolved fluorescence to study proposed conformational transitions in the Ca-ATPase in skeletal sarcoplasmic reticulum (SR). Resonance energy transfer was used to measure distances between the binding sites of 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS) and fluorescein 5-isothiocyanate (FITC) as a function of conditions proposed to affect the enzyme's conformation. When 1.0 +/- 0.15 IAEDANS is bound per Ca-ATPase, most (76 +/- 4%) of the probes have an excited-state lifetime (tau) of 18.6 +/- 0.5 ns, and the remainder have a lifetime of 2.5 +/- 0.9 ns. When FITC is bound to a specific site on each IAEDANS-labeled enzyme, most of the long-lifetime component is quenched into two short-lifetime components, indicating energy transfer that corresponds to two donor-acceptor distances. About one-third of the quenched population has a lifetime tau = 11.1 +/- 2.5 ns, corresponding to a transfer efficiency E = 0.40 +/- 0.07 and a donor-acceptor distance R1 = 52 +/- 3 A. The remaining two-thirds exhibit lifetimes in the range of 1.2-4.2 ns, corresponding to a second distance 31 A less than or equal to R2 less than or equal to 40 A. Addition of Ca2+ (in the micromolar to millimolar range), or vanadate (to produce a phosphoenzyme analogue), had no effect on the donor-acceptor distances. Addition of decavanadate results in the quenching of IAEDANS fluorescence but has no effect on the energy-transfer distance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.     

Development and characterization of green fluorescent protein mutants with altered lifetimes

Multiple-probe fluorescence imaging applications demand an ever-increasing number of resolvable probes, and the use of fluorophores with resolvable fluorescence lifetimes can help meet this demand. Green fluorescent protein (GFP) and its variants have been widely used in spectrally resolved multiprobe imaging, but as yet, there has not been a systematic set of mutants generated with resolvable lifetimes. Therefore, to generate such mutants, we have utilized error-prone PCR and fluorescence lifetime imaging to screen for mutants of UV-excited green fluorescent protein (GFPuv) that exhibit altered fluorescence decay lifetimes. This has resulted in the isolation of GFPuv mutants displaying at least three distinctly different lifetimes in the range of 1.9-2.8 ns. Mutation of Y145 to either histidine or cysteine was found to shift the fluorescence lifetime of GFPuv from 3.03 +/- 0.03 to 2.78 +/- 0.05 ns for the Y145H mutant and to 2.74 +/- 0.05 ns for Y145C. Some of the shorter-lifetime mutants exhibited excitation peaks that were red-shifted relative to their maximal absorption, indicating that the mutations allowed the adoption of additional conformations relative to wtGFPuv. The utility of these mutants for applications in simultaneous imaging and quantification is shown by the ability to quantify the composition of binary mixtures in time-resolved images using a single detector channel. The application of the screening method for generating lifetime mutants of other fluorescent proteins is also discussed.     

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.     

Ultrafast carotenoid-to-chlorophyll singlet energy transfer in the cytochrome b6f complex from Bryopsis corticulans

Ultrafast carotenoid-to-chlorophyll (Car-to-Chl) singlet excitation energy transfer in the cytochrome b(6)f (Cyt b(6)f) complex from Bryopsis corticulans is investigated by the use of femtosecond time-resolved absorption spectroscopy. For all-trans-alpha-carotene free in n-hexane, the lifetimes of the two low-lying singlet excited states, S(1)(2A(g)(-)) and S(2)(1B(u)(+)), are determined to be 14.3 +/- 0.4 ps and 230 +/- 10 fs, respectively. For the Cyt b(6)f complex, to which 9-cis-alpha-carotene is bound, the lifetime of the S(1)(2A(g)(-)) state remains unchanged, whereas that of the S(2)(1B(u)(+)) state is significantly reduced. In addition, a decay-to-rise correlation between the excited-state dynamics of alpha-carotene and Chl a is clearly observed. This spectroscopic evidence proves that the S(2)(1B(u)(+)) state is able to transfer electronic excitations to the Q(x) state of Chl a, whereas the S(1)(2A(g)(-)) state remains inactive. The time constant and the partial efficiency of the energy transfer are determined to be 240 +/- 40 fs and (49 +/- 4)%, respectively, which supports the overall efficiency of 24% determined with steady-state fluorescence spectroscopy. A scheme of the alpha-carotene-to-Chl a singlet energy transfer is proposed based on the excited-state dynamics of the pigments.     

One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins

We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at approximately 440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore.     

Steady-state and picosecond time-resolved fluorescence studies on native and apo seed coat soybean peroxidase.

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase super family. The protein has a very similar 3-dimensional structure with that of horseradish peroxidase (HRP-C). The fluorescence characteristics of the single tryptophan (Trp117) present in SBP and apo-SBP have been studied by steady-state and pico-second time-resolved fluorescence spectroscopy. Fluorescence decay curve of SBP was best described by a four exponential model that gave the lifetimes, 0.035 ns (97.0%), 0.30 ns (2.0%), 2.0 ns (0.8%), and 6.3 ns (0.2%). These lifetime values agreed very well with the values obtained by the model independent maximum entropy method (MEM). The three longer lifetimes that constituted 3% of the fluorophore population in the SBP sample are attributed to the presence of trace quantities of apo-SBP. The pico-second lifetime value of SBP is indicative of efficient energy transfer from Trp117 to heme. From fluorescence resonance energy transfer (FRET) calculations, the energy-transfer efficiency in SBP is found to be relatively higher as compared to HRP-C and is attributed mainly to the higher value of orientation factor, kappa(2) for SBP. Decay-associated spectra of SBP indicated that the tryptophan of SBP is relatively more solvent exposed as compared to HRP-C and is attributed to the various structural features of SBP. Linear Stern-Volmer plots obtained from the quenching measurements using acrylamide gave k(q) = 5.4 x 10(8) M(-1) s(-1) for SBP and 7.2 x 10(8) M(-1) s(-1) for apo-SBP and indicated that on removal of heme in SBP, Trp117 is more solvent exposed.     

Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles

The two-state excited-state proton-transfer process for d-equilenin [d-3-hydroxyestra-1,3,-5(10),6,8-pentaen-17-one] and dihydroequilenin is found to depend both on pH and on proton acceptor concentration. Both the protonated and deprotonated forms of the excited molecule are fluorescent. As is the case for 2-naphthol, the excited-state pKa (pKa*) is substantially lower than the ground-state pKa. Fluorescence decay studies have been performed as a function of emission wavelength in aqueous solutions at pH 6.9 in the presence of acetate anion (0.1 M). At this pH, both back-reaction from the excited-state and ground-state heterogeneity are minimal. A monoexponential decay is found in the blue region of the spectrum and a biexponential decay on the red edge. The lifetimes measured across both regions are constant, with a negative preexponential term, characteristic of an excited-state reaction, evident at longer wavelengths. Decay-associated spectra (DAS), the preexponential terms associated with the measured lifetimes, have been acquired for these aqueous solutions. Equilenin and dihydroequilenin are found to adsorb to dimyristoyllecithin (DML) vesicles. Rates for excited-state proton transfer are greatly reduced when dihydroequilenin adsorbs to vesicles. The accessibility of the bound probe to acetate as a proton acceptor depends on the cholesterol content of the vesicles.     

Time-resolved thermodynamic profiles for CO photolsysis from the mixed valence form of bovine heart cytochrome c oxidase.

Photoacoustic calorimetry has been utilized to probe the thermodynamics accompanying photodissociation of the CO mixed valence form of bovine heart cytochrome c oxidase (COMV CcO). At pH's below 9 photolysis of the COMV CcO results in three kinetic phases with the first phase occurring faster than the time resolution of the instrument (i.e., < approximately 50 ns), a second phase occurring with a lifetime of approximately 100 ns and a third phase occurring with a lifetime of approximately 2 micros. The corresponding volume and enthalpy changes for these processes are: DeltaH1, DeltaV1 = +79 +/- 10 kcal mol(-1), +9 +/- 1 mL mol(-1); DeltaH2, DeltaV2 = -79 +/- 5 kcal mol(-1), -9 +/- 2 mL mol(-1); DeltaH3, DeltaV3 = +54 +/- 7 kcal mol(-1), +8 +/- 1 mL mol(-1). At pH's above 9 only one phase is observed, a prompt phase occurring in < 50 ns. The overall volume change is negligible above pH 9 and the enthalpy change is +29 +/- 5 kcal mol(-1). The data are consistent with the prompt phase being associated with CO-Fe(a3) bond cleavage, CO-CuB+ bond formation, Fe(a3) low-spin to high-spin transition and fast electron transfer (ET) from heme a3 to heme a followed by proton transfer from Glu242 to Arg38 on an approximately 100 ns timescale. The slow phase is likely a combination of CO thermal dissociation from CuB and additional ET between heme a3 to heme a. Interestingly, this phase is not evident above pH 9 suggesting linkage between CO dissociation/ET and the protonation state of a group or groups near the binuclear center.     

Calcium-induced conformational change in fragment 1-86 of factor X

Time-resolved fluorescence of the single tryptophan residue Trp41 in fragment 1-86 of factor X (FX F1-86) is studied using a time-correlated single photon counting technique with synchrotron radiation as the excitation source. Calcium ions are believed to induce a conformational change in the N-termini of the activated factor X and other vitamin K dependent proteins, which is accompanied by a decrease in fluorescence intensity. The titration with calcium yields a sigmoidal fluorescence titration curve with a transition midpoint concentration of 0.44 mM. The wavelength-dependent tryptophan fluorescence decays of the apo-FX F1-86 (in the absence of calcium) and Ca-FX F1-86 are characterized by conventional multiexponential analysis and fluorescence lifetime distribution analysis. In the absence of calcium there are three significant classes of fluorescence lifetimes (ns) that are nearly wavelength independent: 0.55 +/- 0.08 (component A), 2.6 +/- 0.1 (component B), and 5.3 +/- 0.3 (component C). However, their preexponential amplitudes vary with wavelength. The decay associated emission spectra of the individual components show that components B and C contribute over 85% to the total fluorescence for all examined wavelengths. However, in the presence of calcium, the analysis of the time-resolved fluorescence data of Ca-FX F1-86 yields four wavelength-independent lifetimes (ns) of 0.30 +/- 0.09 (component D), 0.65 +/- 0.10 (component A), 2.7 +/- 0.2 (component B), and 5.4 +/- 0.3 (component C). Calcium addition to the apo-FX F1-86 leads to a decrease in the fluorescence intensities of components B and C while their decay times remain unaffected. In Ca-FX F1-86 an additional component D arises that has a decay time of 0.30 ns and that contributes up to 35% to the total fluorescence intensity. A comparison with a previous investigation of prothrombin fragment 1 demonstrates the extensive structural and functional homology between the N termini of prothrombin and factor X(a).     

Time-resolved single tryptophan fluorescence in photoactive yellow protein monitors changes in the chromophore structure during the photocycle via energy transfer

We show from time-resolved fluorescence intensity and depolarization experiments that the fluorescence of the unique tryptophan W119 of PYP is quenched by energy transfer to the 4-hydroxycinnamoyl chromophore. Whereas the intensity decay has a time constant of 0.18 ns in P, the decay in the absence of the cofactor (apo-PYP) has a single exponential lifetime of 4.8 ns. This difference in lifetime with and without acceptor can be explained quantitatively on the basis of energy transfer and the high-resolution X-ray structure of P, which allows an accurate calculation of the kappa2 factor. Fluorescence depolarization experiments with donor and acceptor indicate that both are immobilized so that kappa2 is constant on the fluorescence time scale. Using background illumination from an LED emitting at 470 nm, we measured the time-resolved fluorescence in a photostationary mixture of P and the intermediates I2 and I2'. The composition of the photostationary mixture depends on pH and changes from mainly I2 at low pH to predominantly I2' at high pH. The I2/I2' equilibrium is pH-dependent with a pKa of approximately 6.3. In I2 the lifetime increases to approximately 0.82 ns. This is not due to a change in distance or to the increase in spectral overlap but is primarily a consequence of a large decrease in kappa2. Kappa2 was calculated from the available X-ray structures and decreases from approximately 2.7 in P to 0.27 in I2. This change in kappa2 is caused by the isomerization of the acceptor, which leads to a reorientation of its transition dipole moment. We have here a rare case of the kappa2 factor dominating the change in energy transfer. The fluorescence decay in the light is pH-dependent. From an SVD analysis of the light/dark difference intensity decay at a number of pH values, we identify three species with associated lifetimes: P (0.18 ns), I2 (0.82 ns), and X (0.04 ns). On the basis of the pH dependence of the amplitudes associated with I2 and X, with a pKa of approximately 6.3, we assign the third species to the signaling state I2'. The absorption spectra of the 0.82 and 0.04 ns species were calculated from the pH dependence of their fluorescence amplitudes and of the photostationary light/dark difference absorption spectra. The lambda(max) values of these spectra (372 and 352 nm) identify the 0.82 and 0.04 ns components with I2 and I2', respectively, and validate the fluorescence data analysis. The mutant E46Q allows a further test of the energy transfer explanation, since lowering the pH in the dark leads to a bleached state with an increased spectral overlap but without the isomerization-induced decrease in kappa2. The measured lifetime of 0.04 ns is in excellent agreement with predictions based on energy transfer and the X-ray structure.     

Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria is an obligate dimer and does not form a stable complex with the Ca(2+)-discharged photoprotein clytin

Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Fo?rster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.