Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.     

Correlation between internal motion and emission kinetics of tryptophan residues in proteins

Time-resolved fluorescence anisotropy measurements of tryptophan residues were carried out for 44 proteins. Internal rotational motion with a sub-nanosecond correlation time (0.9 +/- 0.6 ns at 10 degrees C) was seen in a large number of proteins, though its amplitude varied from protein to protein. It was found that tryptophan residues which were almost fixed within a protein had either a long (greater than 4 ns) or short (less than 2 ns) fluorescence lifetime, whereas a residue undergoing a large internal motion had an intermediate lifetime (1.5-3 ns). It is suggested that the emission kinetics of a tryptophan residue is coupled with its internal motion. In particular, an immobile tryptophan residue emitting at long wavelength was characterized by a long lifetime (greater than 4 ns). It appears that a tryptophan residue fixed in a polar region has little chance of being quenched by neighboring groups.     

Time-resolved fluorescence and anisotropy decay of the tryptophan in adrenocorticotropin-(1-24)

The direct time-resolved fluorescence anisotropy of the single tryptophan residue in the polypeptide hormone adrenocorticotropin-(1-24) (ACTH) and the fluorescence decay kinetics of this residue (Trp-9) are reported. Two rotational correlation times are observed. One, occurring on the subnanosecond time scale, reflects the rotation of the indole ring, and the other, which extends into the nanosecond range, is dominated by the complex motions of the polypeptide chain. The fluorescence lifetimes of the single tryptophan in glucagon (Trp-25) and the 23-26 glucagon peptide were also measured. In all cases the fluorescence kinetics were satisfied by a double-exponential decay law. The fluorescence lifetimes of several tryptophan and indole derivatives and two tryptophan dipeptides were examined in order to interpret the kinetics. In close agreement with the findings of Szabo and Rayner [Szabo, A. G., & Rayner, D. M. (1980) J. Am. Chem. Soc. 102, 554-563], the tryptophan zwitterion exhibits emission wavelength dependent double-exponential decay kinetics. At 320 nm tau 1 = 3.2 ns and tau 2 = 0.8 ns, with alpha 1 = 0.7 and alpha 2 = 0.3. Above 380 nm only the 3.2-ns component is observed. By contrast the neutral derivative N-acetyltryptophanamide has a single exponential decay of 3.0 ns. The multiexponential decay kinetics of the polypeptides are discussed in terms of flexibility of the polypeptide chain and neighboring side-chain interactions.     

Rotational freedom of tryptophan residues in proteins and peptides

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.     

Frequency-domain fluorescence studies of an extracellular metalloproteinase of Staphylococcus aureus

A frequency-domain fluorescence study of calcium-binding metalloproteinase from Staphylococcus aureus has shown that this two-tryptophan-containing protein exhibits a double-exponential fluorescence decay. At 10 degrees C in 0.05 M Tris-HCl buffer (pH 9.0) containing 10 mM CaCl2, fluorescence lifetimes of 1.2 and 5.1 ns are observed. Steady-state and frequency-domain solute-quenching studies are consistent with the assignment of the two lifetimes to the two tryptophan residues. The tryptophan residue characterized by a shorter lifetime has a maximum of fluorescence emission at about 317 nm and the second one exhibits a maximum of its emission at 350 nm. These two residues contribute almost equally to the protein's fluorescence. These results, as well as fluorescence-quenching studies using KI and acrylamide as a quencher, indicate that in calcium-loaded metalloproteinase, the tryptophan residue characterized by the shorter lifetime is extensively buried within the protein. The second residue is exposed on the surface of the protein. The tryptophan residues of metalloproteinase have acrylamide dynamic-quenching rate constants, kq values, of 2.3 and 0.26 X 10(9) M-1 X s-1 for the exposed and buried residue, respectively. A study of the temperature dependence of the fluorescence lifetime for the two tryptophan components gives activation energies, Ea values, for thermal quenching of 1.8 and 2.2 kcal/mol for the buried and the exposed residue, respectively. Dissociation of Ca2+ from the protein causes a change in the protein's structure, as can be judged from dramatic changes which occur in the fluorescence properties of the buried tryptophan residue. These changes include an approx. 13 nm red-shift in the maximum of the fluorescence emission and an increase in the acrylamide-quenching rate constant, and they indicate that the removal of Ca2+ results in an increase in the exposure and the polarity of the microenvironment of this 'blue' residue.     

Time resolved spectroscgpy of tryptopkyl fluorescence of yeast 3-phosphoglycerate kinase

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime τ₁= 0.6 ns and a maximum fluorescence wavelength λ_2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ₂₁= 3.1 ns and τ₂₂= 7.0 ns (λ_2max= 338 nm). In agreement with the attribution of τ₂₁and τ₃₂ to the same tryptophan residue, the ratio β = C₂₁/C₂₂ of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally tc the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay times and a neighbouring protein chemical group. The intrinsic pK of this group and the equilibrium constant of the irternal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.     

Investigation of the structural determinants of the intrinsic fluorescence emission of the trp repressor using single tryptophan mutants.

The fluorescence decay properties of wild-type trp repressor (TR) have been characterized by carrying out a multi-emission wavelength study of the frequency response profiles. The decay is best analyzed in terms of a single exponential decay near 0.5 ns and a distribution of lifetimes centered near 3-4 ns. By comparing the recovered decay associated spectra and lifetime values with the structure of the repressor, tentative assignments of the two decay components recovered from the analysis to the two tryptophan residues, W19 and W99, of the protein have been made. These assignments consist of linking the short, red emitting component to emission from W99 and most of the longer bluer emitting lifetime distribution to emission from W19. Next, single tryptophan mutants of the repressor in which one of each of the tryptophan residues was substituted by phenylalanine were used to confirm the preliminary assignments, inasmuch as the 0.5-ns component is clearly due to emission from tryptophan 99, and much of the decay responsible for the recovered distribution emanates from tryptophan 19. The data demonstrate, however, that the decay of the wild-type protein is not completely resolvable due both to the large number of components in the wild-type emission (at least five) as well as to the fact that three of the five lifetime components are very close in value. The fluorescence decay of the wild-type decay is well described as a combination of the components found in each of the mutants. However, whereas the linear combination analysis of the 15 data sets (5 from the wild-type and each mutant) yields a good fit for the components recovered previously for the two mutants, the amplitudes of these components in the wild-type are not recovered in the expected ratios. Because of the dominance of the blue shifted emission in the wild-type protein, it is most likely that subtle structural differences in the wild-type as compared with the mutants, rather than energy transfer from tryptophan 19 to 99, are responsible for this failure of the linear combination hypothesis.     

Fluorescence studies of phosphoribulokinase, a light-regulated, chloroplastic enzyme

Recent characterization of spinach phosphoribulokinase has revealed that the homodimeric molecule contains only two tryptophans per 44-kDa subunit. We have performed steady-state and frequency domain studies of the intrinsic fluorescence of this protein. The fluorescence properties reflect contributions from both types of tryptophan residues. One of these appears to be relatively exposed to solvent and the quencher, acrylamide; fluoresce with a lambda max of 345 nm; decay with a fluorescence lifetime of 6.3 ns; have a relatively red-shifted absorption spectrum; and have a certain degree of independent motional freedom, with respect to the protein. The other tryptophan residue appears to be more buried; fluoresce with lambda max of 325 nm; have a lifetime of 1.7 ns; have a relatively blue-shifted absorption spectrum; and not to enjoy independent motional freedom. On comparison of phase-resolved spectral data and solute quenching data, we suggest that resonance energy transfer between the blue and red tryptophan residues may occur. We also describe the strategy of simultaneously fitting Stern-Volmer quenching data collected at two emission wavelengths.     

A time-resolved fluorescence study of human copper-zinc superoxide dismutase

The intrinsic fluorescence decay of human Cu,Zn superoxide dismutase was measured by frequency-domain techniques. The protein consists of two subunits, each containing one tryptophan and no tyrosine residues. Using a synchrotron radiation source, which allows facile selection of the excitation wavelength, the dependence of the emission decay upon excitation was studied. No significant excitation wavelength effects were found. The two tryptophans contained in the dimer, although fully equivalent and exposed to solvent, showed a fluorescence decay that cannot be described by a single lifetime. Either two lifetimes, or one Lorentzian-shaped continuous distribution of lifetimes, are needed to obtain a good fit. Under identical experimental conditions, control experiments showed that N-acetyltryptophanamide, an analogue of tryptophanyl residues in proteins, decays with a single lifetime. The heterogeneous decay of tryptophan fluorescence in superoxide dismutase is interpreted as due to the presence of static and/or dynamic conformers in the protein that decay with different lifetimes. The two models of discrete lifetimes and continuous distribution of lifetimes are discussed with reference to measurements on holo- and apo-human superoxide dismutase.     

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 dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.     

Photophysics of tryptophan in bacteriophage T4 lysozymes

Bacteriophage T4 lysozyme contains three tryptophan residues in distinct environments. Lysozymes with one or two of these residues replaced by tyrosine are used to characterize the photophysics of tryptophan in these individual sites. The fluorescence spectra, average lifetimes, and quantum yields of these three single-tryptophan variants are understandable in terms of the neighboring residues. The emission spectra and radiative lifetimes are found to be the same for all three species while the quantum yield and decay kinetics are quite distinct. The variation of the average nonradiative rate constant is correlated with neighboring quenching groups. Quenching by I- correlates with exposure of the tryptophan residue based on the crystal structure. Complex behavior is observed for the time dependence of the fluorescence decay in all three cases, including that of the immobile tryptophan-138 residue. The complexity of the fluorescence decay is ascribed to heterogeneity in the nonradiative rate constant among microstates. Energy transfer between tryptophan residues is inferred to occur from comparison of the quantum yields of the two-tryptophan and single-tryptophan proteins and is discussed in terms of the F?rster mechanism.     

Nanosecond pulse fluorometry of conformational change in phenylalanine hydroxylase associated with activation

Conformational change in rat liver phenylalanine hydroxylase associated with activation by phenylalanine or N-(1-anilinonaphth-4-yl)maleimide was investigated by measuring fluorescence spectra and fluorescence lifetimes of tryptophanyl residues as well as the probe fluorophore conjugated with SH groups of the hydroxylase. The fluorescence spectrum of tryptophan exhibited its maximum at 342 nm. It shifted by 8 nm toward longer wavelength accompanied by an increase in its intensity, by preincubation with 1 mM phenylalanine. The fluorescence intensity of tryptophan increased by 36% upon the activation. On the other hand, the binding of (6R)-L-erythro-tetrahydrobiopterin, a natural cofactor of the enzyme, induced a decrease in the fluorescence intensity by 79% without a shift of the maximum wavelength. The fluorescence lifetime of tryptophan of phenylalanine hydroxylase exhibited two components with lifetimes of 1.7 and 4.1 ns. The values of the lifetimes changed to 1.4 and 5.6 ns, respectively, upon the activation. It is considered that the change in the longer lifetime is correlated with the shift of the emission peak upon the activation. The values of both the lifetimes decreased to 0.64 and 3.6 ns upon the binding of (6R)-L-erythro-tetrahydrobiopterin, which is coincident with the decrease in the fluorescence intensity. Conjugation of N-(1-anilinonaphth-4-yl)maleimide with SH of phenylalanine hydroxylase brought about a decrease in both the fluorescence intensity and the value of the shorter lifetime of the tryptophanyl residues, while the longer lifetime remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Constrained analysis of fluorescence anisotropy decay:application to experimental protein dynamics

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.     

Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.     

Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization.

The fluorescence decay of apoazurin derived from Pseudomonas aeruginosa is monoexponential. By this criterion the population of molecules of apoazurin is homogeneous. The emission anisotropy factor and the absorption anisotropy factor at the red edge of the absorption band assume similar values, showing that the tryptophan residue in apoazurin has the same asymmetric environment both in the ground and excited states. This finding suggests tight packing of the protein at the tryptophan environment. Native azurin does not decay monoexponentially. Moreover, comparison between the quantum yield calculated from the decay kinetics and the one measured directly shows that the majority of the azurin molecules are not fluorescent. There is thus variability in the structure of azurin molecules with an equilibration time that is longer than the fluorescence lifetime. Different asymmetric environment was found for the tryptophan residue in oxidized and reduced holoprotein and in apoazurin, as studied by the circular polarization of the fluorescence. D(2)O increases the fluorescence lifetime of apoazurin by 6 percent, compared to the lifetime in H(2)O solution; therefore water molecules may have access to the tryptophan residue, though the latter is situated in a hydrophobic environment.     

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).     

Tryptophanyl fluorescence lifetime distribution of hyperthermophilic beta-glycosidase from molecular dynamics simulation: a comparison with the experimental data

A molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for beta-glycosidase from the hyperthermophilic bacterium Solfolobus sulfotaricus (Sbeta gly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of Sbeta gly results from a bimodal distribution of fluorescence lifetimes with a short-lived component centered at 2.5 ns and a long-lived one at 7.4 ns (Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71-79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern-Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of Sbeta gly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition Sbeta gly evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side-chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results.