Determination of the triplet state energies of a series of conjugated porphyrin oligomers.

We report a systematic study of the photophysical parameters relevant to photodynamic therapy (PDT) by a new type of sensitizers, conjugated porphyrin oligomers. Due to the strong nonlinear properties of oligomers containing 2, 4 and 8 porphyrin units, these molecules are attractive candidates for PDT via multiphoton excitation. The triplet state energy levels for all molecules have been determined by the triplet quenching method, phosphorescence measurements and DFT calculations. We find that the triplet energies of all the oligomers are sufficient to generate singlet oxygen, >94 kJ mol(-1). However, low singlet oxygen quantum yields are observed for the tetramer and the octamer, as compared to the conjugated dimer and monomeric porphyrin, reflecting the decrease in triplet yield. Thus the conjugated porphyrin dimer is the most promising core structure for PDT applications via multiphoton excitation.     

Meso-tetraphenylporphyrin in liposomes as a suitable photosenzitizer for photodynamic therapy of tumors.

The suitability of a liposomal form of hydrophobic nonsulfonated meso-tetraphenyl porphyrin (TPP) for the photodynamic therapy of tumors was investigated. TPP was solubilized in small unilamellar lipid vesicles prepared by extrusion on a LIPOSOFAST apparatus. These samples were studied by laser-excited time resolved luminescence and triplet-triplet absorption spectroscopy. In this lipid environment TPP was still an efficient singlet oxygen producer, as indicated by the characteristic singlet oxygen phosphorescence at 1270 nm in D2O, when excited with a 28 ns laser pulse at 412 nm. Moreover, unlike with sulfonated TPP (TPPS4), liposomal TPP showed the reduced decay rates of TPP triplet-states with the increasing time of pre-illumination by a Xenon lamp. This was shown in an indirect way, based upon the appearance of a second component of the luminescence decay at 1270 nm in D2O; and by direct TPP triplet state monitoring, detecting triplet-triplet absorption at 440 nm in H2O. The deactivation of higher triplet states was delayed upon pre-illumination. This reflects an irreversible interaction of singlet oxygen with membrane lipids, thus demonstrating the potential of the liposomal form of TPP to efficiently disintegrate tumor cell membranes and to be a suitable preparation for the photodynamic therapy.     

Phosphorescence study of chlorophyll d photophysics. Determination of the energy and lifetime of the photo-excited triplet state. Evidence of singlet oxygen photosensitization

Chlorophyll d (Chl d) is the major pigment in both photosystems (PSI and II) of the cyanobacterium Acaryochloris marina, whose pigment composition represents an interesting alternative in oxygenic photosynthesis. While abundant information is available relative to photophysical properties of Chl a , the understanding of Chl d photophysics is still incomplete. In this paper, we present for the first time a characterization of Chl d phosphorescence, which accompanies radiative deactivation of the photoexcited triplet state of this pigment. Reliable information was obtained on the energy and lifetime of the Chl d triplet state in frozen solutions at 77?K using diethyl ether and aqueous dispersions of Triton X100 as solvents. It is shown that triplet Chl d is effectively populated upon photoexcitation of pigment molecules and efficiently sensitizes singlet oxygen phosphorescence in aerobic solutions under ambient conditions. The data obtained are compared with the previous results of the phosphorescence studies of Chl a and Pheo a, and their possible biological implications are discussed.     

Quenching of fluorescence by triplet excited states in chloroplasts

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5-4.0 mus duration dye laser flash of varying intensities as an excitation source. The yield (phi) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectoscopy at 515 nm, while the yield phi following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10-12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from phi utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (greater than 10(16) photon . cm-2) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1-2 mus after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules. At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse. A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a single-triplet bimolecular rate constant of gammast = 10(-8) cm3 . s-1, the following observations can be accounted for: (1) the rapid initial drop in phi and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.     

Excited states of tryptophan in cod parvalbumin. Identification of a short-lived emitting triplet state at room temperature.

The fluorescence and phosphorescence spectra of model indole compounds and of cod parvalbumin III, a protein containing a single tryptophan and no tyrosine, were examined in the time scale ranging from subnanoseconds to milliseconds at 25 degrees C in aqueous buffer. For both Ca- bound and Ca-free parvalbumin and for model indole compounds that contained a proton donor, a phosphorescent species emitting at 450 nm with a lifetime of approximately 20-40 ns could be identified. A longer-lived phosphorescence is also apparent; it has approximately the same absorption and emission spectrum as the short-lived triplet molecule. For Ca parvalbumin, the decay of the long-lived triplet tryptophan is roughly exponential with a lifetime of 4.7 ms at 25 degrees C whereas for N-acetyltryptophanamide in aqueous buffer the decay lifetime was 30 microseconds. In contrast, the lifetime of the long-lived tryptophan species is much shorter in the Ca-free protein compared with Ca parvalbumin, and the decay shows complex nonexponential kinetics over the entire time range from 100 ns to 1 ms. It is concluded that the photochemistry of tryptophan must take into account the existence of two excited triplet species and that there are quenching moieties within the protein matrix that decrease the phosphorescence yield in a dynamic manner for the Ca-depleted parvalbumin. In contrast, for Ca parvalbumin, the tryptophan site is rigid on the time scale of milliseconds.     

Room temperature phosphorescence of Trp-314 as a monitor of subunit communications in alcohol dehydrogenase from horse liver

The phosphorescence properties of liver alcohol dehydrogenase from horse were characterized at limiting concentrations of coenzyme and coenzyme analogues. The emission decay kinetics of Trp-314 in strong, slowly exchanging, ternary complexes with NADH/isobutyramide, NAD/pyrazole, and NADH/dimethyl sulfoxide displays a markedly nonexponential character. The analysis of decay components over the saturation curve reveals that the phosphorescence from singly bound protein molecules has a lifetime from 1 to 1.3 s, which is 2-3 times larger than observed with fully bound and unliganded enzyme. The remarkably tighter configuration reported by the triplet probe for the coenzyme-binding domain in half-saturated macromolecules is not exclusive of strongly inhibited ternary complexes. Measurements on binary complexes with NADH, ADPR, and the inactive coenzyme analogue 1,4,5,6-tetrahydronicotinamide adenine dinucleotide confirm that binding of the ligand to one subunit has qualitatively the same influence on protein structure. If the lifetime of Trp-314 provides clear evidence for an appreciable change in conformation at half-binding that is apparently triggered by the ADPR fragment of the coenzyme, such communication between subunits does not lead to allosteric phenomena in coenzyme binding.     

Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements.

This study describes the use of two-photon excitation phosphorescence lifetime measurements for quantitative oxygen determination in vivo. Doubling the excitation wavelength of Pd-porphyrin from visible light to the infrared allows for deeper tissue penetration and a more precise and confined selection of the excitation volume due to the nonlinear two-photon effect. By using a focused laser beam from a 1,064-nm Q-switched laser, providing 10-ns pulses of 10 mJ, albumin-bound Pd-porphyrin was effectively excited and oxygen-dependent decay of phosphorescence was observed. In vitro calibration of phosphorescence lifetime vs. oxygen tension was performed. The obtained calibration constants were kq = 356 Torr(-1) x s(-1) (quenching constant) and tau0 = 550 micros (lifetime at zero-oxygen conditions) at 37 degrees C. The phosphorescence intensity showed a squared dependency to the excitation intensity, typical for two-photon excitation. In vivo demonstration of two-photon excitation phosphorescence lifetime measurements is shown by step-wise PO2 measurements through the cortex of rat kidney. It is concluded that quantitative oxygen measurements can be made, both in vitro and in vivo, using two-photon excitation oxygen-dependent quenching of phosphorescence. The use of two-photon excitation has the potential to lead to new applications of the phosphorescence lifetime technique, e.g., noninvasive oxygen scanning in tissue at high spatial resolution. To our knowledge, this is the first report in which two-photon excitation is used in the setting of oxygen-dependent quenching of phosphorescence lifetime measurements.     

Kinetics of singlet oxygen photosensitization in human skin fibroblasts

The roles played by singlet oxygen (¹O₂) in photodynamic therapy are not fully understood yet. In particular, the mobility of ¹O₂ within cells has been a subject of debate for the last two decades. In this work, we report on the kinetics of ¹O₂ formation, diffusion, and decay in human skin fibroblasts. ¹O₂ has been photosensitized by two water-soluble porphyrins targeting different subcellular organelles, namely the nucleus and lysosomes, respectively. By recording the time-resolved near-IR phosphorescence of ¹O₂ and that of its precursor the photosensitizer's triplet state, we find that the kinetics of singlet oxygen formation and decay are strongly dependent on the site of generation. ¹O₂ photosensitized in the nucleus is able to escape out of the cells while ¹O₂ photosensitized in the lysosomes is not. Despite showing a lifetime in the microsecond time domain, ¹O₂ decay is largely governed by interactions with the biomolecules within the organelle where it is produced. This observation may reconcile earlier views that singlet oxygen-induced photodamage is highly localized, while its lifetime is long enough to diffuse over long distances within the cells.     

Quenching of fluorescence by triplet excited states in chloroplasts

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5–4.0 μs duration dye laser flash of varying intensities as an excitation source. The yield (Ф) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectroscopy at 515 nm, while the yield Ф following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10–12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from Ф utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (10¹⁶ photon · cm⁻²) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1–2 μs after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules.

At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse.

A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a singlet-triplet bimolecular rate constant of γ_st = 10⁻⁸ cm³ · s⁻¹, the following observations can be accounted for: (1) the rapid initial drop in Ф and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.     

Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.

The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.     

Singlet molecular oxygen in photobiochemical systems: IR phosphorescence studies.

Singlet molecular oxygen (1O2) is one of the most active intermediates involved in photosensitized oxygenation reactions in chemical and biological systems. Deactivation of singlet oxygen is accompanied by infrared phosphorescence (1270 nm) which is widely employed for 1O2 detection and study. This review considers techniques for phosphorescence detection, phosphorescence spectra, quantum yields and kinetics under laser excitation, the radiative and real 1O2 lifetimes in organic solvents and water, 1O2 quenching by biomolecules, and estimation of singlet oxygen lifetimes, diffusion lengths and phosphorescence quantum yields in blood plasma, cell cytoplasm, erythrocyte ghosts, retinal rod outer segments and chloroplast thylakoids. The experiments devoted to 1O2 phosphorescence detection in photosensitizer-containing living cells are discussed in detail. Information reviewed is important for understanding the mechanisms of photodestruction in biological systems and various applied problems of photobiology and photomedicine.     

Tryptophan phosphorescence of G-actin and F-actin

The tryptophan phosphorescence spectrum, intensity and decay kinetics of G-actin and F-actin were measured over a temperature range of 140-293 K. The fine structure in the phosphorescence spectra at low temperature, with O,O vibrational bands centered at 405 nm and 415.5 nm for both species, reveals a marked heterogeneity of the chromophore environment. The thermal quenching profile distinguishes these sites in terms of their flexibility, and shows that probably only one of the four tryptophan residues is still phosphorescent at ambient temperature due to its location in a relatively rigid buried core. Although some differences are demonstrated between G-actin and F-actin at low temperature, the identity of the triplet lifetime at ambient temperature strongly supports the notion that the conformation of the macromolecule is largely unaffected by polymerization. Preliminary phosphorescence anisotropy measurements demonstrate both the occurrence of singlet-singlet energy transfer among tryptophan residues and a strong immobilization of actin in the polymerized state.     

Protic solvent effects on the photophysical properties of O=Ti(IV)TSPP: photoinduced electron transfer.

The photophysical properties of oxotitanium(IV)meso-tetra(4-sulfonatophenyl) porphyrin (O=Ti(IV)TSPP) have been investigated in water and methanol by laser spectroscopic techniques. The fluorescence emission spectrum of O=Ti(IV)TSPP in methanol exhibits two strong emission bands at 610 and 670 nm at room temperature with the decay time of ca. 310 +/- 10 ps and the rise time shorter than 30 ps, in contrast to the extremely weak emission with the decay time of ca. 27 +/- 4 ps in water, indicating that the fluorescence emissive states are different in the two solvents as supported by the solvent dependences of the excitation spectrum. The transient Raman spectra of O=Ti(IV)TSPP in water has been observed to exhibit a remarkable enhancement of phenyl-related mode at 1599 cm(-1), while in methanol, the Raman frequencies of the porphyrin skeletal modes (upsilon2 and upsilon4) are down-shifted without any apparent enhancement of the phenyl-related mode, indicating different interactions of the two solvents with the excited O=Ti(IV)TSPP. These Raman studies reveal that methanol molecule interacts with the photoexcited O=Ti(IV)TSPP more strongly than water, forming the exciplex, O=Ti(IV)TSPP(MeOH)*, suggesting that the two different emissive states are the singlet Franck-Condon state and the exciplex state in methanol and water, respectively. A broad triplet transient absorption of O=Ti(IV)TSPP has been also observed at 480 nm in water as well as in methanol, which is decreased upon addition of methyl viologen (MV2+) with appearance of a new absorption band at 620 nm. This indicates that the photoinduced electron transfer (PET) takes place from the porphyrin to MV2+ in both solvents. The kinetic analysis of the transient absorption band exhibits the PET rate constants of 4.76 x 10(5) s(-1) and 3,03 x 10(4) s(-1) in methanol and water, respectively. All these results infer that the PET takes place from the (d,pi) CT state and the triplet state of the excited porphyrin in methanol and water, respectively.     

Spectral and kinetic parameters of phosphorescence of triplet chlorophyll a in the photosynthetic apparatus of plants

Spectral and kinetic parameters and quantum yield of IR phosphorescence accompanying radiative deactivation of the chlorophyll a (Chl a) triplet state were compared in pigment solutions, greening and mature plant leaves, isolated chloroplasts, and thalluses of macrophytic marine algae. On the early stages of greening just after the Shibata shift, phosphorescence is determined by the bulk Chl a molecules. According to phosphorescence measurement, the quantum yield of triplet state formation is not less than 25%. Further greening leads to a strong decrease in the phosphorescence yield. In mature leaves developing under normal irradiation conditions, the phosphorescence yield declined 1000-fold. This parameter is stable in leaves of different plant species. Three spectral forms of phosphorescence-emitting chlorophyll were revealed in the mature photosynthetic apparatus with the main emission maxima at 955, 975, and 995 nm and lifetimes ～1.9, ～1.5, and 1.1–1.3 ms. In the excitation spectra of chlorophyll phosphorescence measured in thalluses of macrophytic green and red algae, the absorption bands of Chl a and accessory pigments — carotenoids, Chl b, and phycobilins — were observed. These data suggest that phosphorescence is emitted by triplet chlorophyll molecules that are not quenched by carotenoids and correspond to short wavelength forms of Chl a coupled to the normal light harvesting pigment complex. The concentration of the phosphorescence-emitting chlorophyll molecules in chloroplasts and the contribution of these molecules to chlorophyll fluorescence were estimated. Spectral and kinetic parameters of the phosphorescence corresponding to the long wavelength fluorescence band at 737 nm were evaluated. The data indicate that phosphorescence provides unique information on the photophysics of pigment molecules, molecular organization of the photosynthetic apparatus, and mechanisms and efficiency of photodynamic stress in plants.     

Triplet-state kinetics of Zn-porphyrin cytochrome c in micellar media. Measurement of intermicellar exchange rates

The interactions of protein molecules with surfactant assemblies in aqueous and hydrocarbon media have been studied via the triplet-state kinetics of Zn-porphyrin cytochrome c in solutions containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] or a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. In aqueous solution, the observed triplet state decay is single exponential with a lifetime of 8 ms. In aqueous solutions of AOT and in AOT-reversed micellar solutions, biexponential triplet state decays were observed, indicating that interactions between the surfactant and the protein occur, resulting in a change in protein conformation near the porphyrin ring. In CTAB-reversed micellar solutions, quenching of the Zn-porphyrin cytochrome c triplet state by ferricyanide and methyl viologen was studied. Because the quenching is exchange-limited under the conditions used, the exchange rate constants for the water pools can be obtained from these experiments. The observed exchange rate constants are in the range (1-5) x 10(7) M-1 S-1, depending on the water content of the reversed micelles and on the type of quencher used. These values are three orders of magnitude lower than the calculated collision rate of the reversed micelles.     

Phosphorescent analysis of lipid peroxidation products in isolated human erythrocyte membranes

The room temperature phosphorescence of lipid peroxidation products in the composition of isolated human erythrocyte membranes was registered, and its kinetic parameters were determined. The excitation and emission spectra of phosphorescence of lipid peroxidation products in the composition of erythrocyte membranes at 0 degree C measured. The nature of lipid peroxidation products possessing the phosphorescencing capacity was discussed. Based on the analysis of temperature dependences of the intensity and lifetimes of phosphorescence of lipid peroxidation products in the range -2 divided by 26 degrees C, it is concluded that the deactivation of excited triplet states of lipid chromophores was realized by the dynamic type.     

Interaction of electron acceptors with the excited triplet state of Zn cytochrome c

The decay rate of the excited triplet state of Zn cytochrome c was enhanced by electron acceptors including methyl viologen and ferric complexes of cyanide, oxalate, EDTA and cytochrome c at room temperature. Ferrous compounds were several orders of magnitude less effective than the respective ferric form in quenching the phosphorescence. In the presence of ferricytochrome c and ferricyanide the semilogarithmic plots of the decay curve showed an anomalous decay profile in which the rate of interaction appeared to accelerate after excitation. One explanation is that the quenching process was accelerated by a conformational change of the polypeptide chain around the excited triplet state porphyrin. Another explanation is that quenching occurs via an intermediate.     

The balance between charge transfer and non-charge transfer pathways in the sensitization of singlet oxygen by pi pi* triplet states.

A charge transfer (CT) channel and a non-CT deactivation channel, both leading to formation of O(2)((1)Sigma (g)(+)), O(2)((1) Delta(g)) and O(2)((3)Sigma(g)(-)), compete in the quenching of triplet states by O(2). Recent studies by our group demonstrated that these channels are described by rather simple and general quantitative relations. In the present paper we use the detailed kinetic data on the quenching by O(2) of pi pi* triplet sensitizers of three homologous aromatic series in CCl(4) to derive a parameter, which describes the balance between CT and non-CT deactivation. This quantity, p(CT), is the relative contribution of CT mediated deactivation and is easily calculated for a sensitizer of known triplet energy from its quenching rate constant. The parameter p(CT) quantitatively describes the balance between both deactivation channels without requiring any knowledge of oxidation potentials. It is shown how the variation of p(CT) influences the efficiencies and the rate constants of O(2)((1)Sigma(g)(+)), O(2)((1)Delta(g)) and O(2)((3)Sigma(g)(-)) formation in the quenching process.     

Effect of heavy water on protein flexibility

The effects of heavy water (D(2)O) on internal dynamics of proteins were assessed by both the intrinsic phosphorescence lifetime of deeply buried Trp residues, which reports on the local structure about the triplet probe, and the bimolecular acrylamide phosphorescence quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results obtained with several protein systems (ribonuclease T1, superoxide dismutase, beta-lactoglobulin, liver alcohol dehydrogenase, alkaline phosphatase, and apo- and Cd-azurin) demonstrate that in most cases D(2)O does significantly increase the rigidity the native structure. With the exception of alkaline phosphatase, the kinetics of the structure tightening effect of deuteration are rapid compared with the rate of H/D exchange of internal protons, which would then assign the dampening of structural fluctuations in D(2)O to a solvent effect, rather than to stronger intramolecular D bonding. Structure tightening by heavy water is generally amplified at higher temperatures, supporting a mostly hydrophobic nature of the underlying interaction, and under conditions that destabilize the globular fold.     

Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain

The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.