Fluorescence lifetime and spectral study of the acid expansion of bovine serum albumin

The fluorescence lifetimes of the tryptophan residues of bovine serum albumin were measured in the native and acid-expanded conformation. A three-exponential process is required to fit the fluorescence decay data. The results are interpreted empirically in terms of two emitting species. The emission at longer wavelength (360 nm) has slower rates of decay than that at shorter wavelength (325 nm). For both emitting species the average lifetime decreases when the N-F transition occurs and shortens further when the protein expands. Rotational correlation times, derived from the decay of the fluorescence anisotropy of the tryptophan residues, suggest that longer emission wavelengths are associated with somewhat shorter correlation times. There is no certain indication of any independent motion of the tryptophans in any conformation, although some very fast process, perhaps Raman scattering, appears to occur. On acid expansion the long correlation times decrease to around 10 ns in the fully expanded form. Static quenching experiments using I- or acrylamide suggest a greater average exposure of the tryptophans when the protein is most greatly expanded. This is despite the fact that the fluorescence emission maximum shifts to shorter wavelength under these conditions. Also, there is no difference in accessibility to quenching between the longer and shorter wavelength emissions.     

Hydrophobic surfaces of tubulin probed by time-resolved and steady-state fluorescence of nile red

Binding of Nile Red to tubulin enhances and blue-shifts fluorescence emission to about 623 nm with a "shoulder" around 665 nm. Binding is reversible and saturable with an apparent Kd of approximately 0.6 microM. Nile Red does not alter tubulin polymerization, and polymerization in 2-(N-morpholino)ethanesulfonic acid (Mes) buffer does not alter the spectrum of the Nile Red-tubulin complex. In contrast, polymerization in glutamate buffer results in a red shift, reduction of intensity, and a decrease in lifetime, suggesting an increase in "polarity" of the binding environment. Lifetimes of 4.5 and 0.6 ns fluorescence in Mes buffer are associated with the 623-nm peak and the 665-nm shoulder, respectively. Indirect excitation spectra for these components are distinct and the 4.5-ns component exhibits tryptophan to Nile Red energy transfer. Acrylamide quenching yields linear Stern-Volmer plots with unchanged lifetimes, indicating static quenching. Apparent quenching constants are wavelength-dependent; global analysis reveals a quenchable component corresponding to the 4.5 ns component and an "unquenchable" component superposing the 0.6-ns spectrum. Analysis of anisotropy decay required an "associative" model which yielded rotational correlation times of greater than 50 ns for the 4.5-ns lifetime and 0.3 ns for the 0.6-ns lifetime. Dilution of tubulin in Mes results in an apparent red shift of emission without lifetime changes, due only to loss of the 623-nm component. These data are reconciled in terms of a model with two binding sites on the tubulin dimer. The more "nonpolar" site is located in a region of subunit-subunit contact which accounts for the fluorescence changes upon dilution; this permits estimation of a subunit dissociation constant of 1 microM.     

Conformation of adenosine deaminase in complexes with inhibitors: application of selective quenching of fluorescence emission

The effect of inhibitors, 1-deazaadenosine (1-dAdo) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), on the conformation of adenosine deaminase was studied using the method of selective quenching of fluorescence emission by acrylamide, I- and Cs+. Both in free adenosine deaminase and in its complexes with the inhibitors, the wavelength maxima and half-width of the emission characterize the environment of fluorescing tryptophan residues in adenosine deaminase as weak polar with limited access to solvent. The formation of complexes with the ground state inhibitors used did not quench or change the main emission characteristics of tryptophan fluorescence in adenosine deaminase. Small blue shifts of emission maxima were observed upon quenching in all three samples. The Stern-Volmer parameters of tryptophan fluorescence quenching by acrylamide were not essentially influenced by complex formation of the enzyme with the inhibitors: in general, the folding of the enzyme molecule in the complexes is not perturbed. On the contrary, the emission quenching by charged heavy ions, I- and Cs+, in the complexes was hindered in comparison with free adenosine deaminase. In the complex with 1-deazaadenosine, the parameters for quenching by both ions evidence the essential worsening of their interaction with tryptophans. In the complex with erythro-9-(2-hydroxy-3-nonyl)adenine, along with the worse quenching by I-, complete prohibition of quenching by Cs+ was observed. These data indicate that the local environments of fluorescing tryptophan residues is substantially distorted compared with free adenosine deaminase, which leads to their screening from charged heavy ions.     

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.     

Fluorescence studies with malate dehydrogenase from Bradyrhizobium japonicum 3I1B-143 bacteroids: a two-tryptophan containing protein

A number of fluorescence studies, both of trp residues and bound NADH, have been reported for porcine malate dehydrogenase (MDH). The large number of trp residues (six) complicates the interpretation of some studies. To circumvent this we have performed studies with a two-tryptophan (per subunit) MDH from Bradyrhizobium japonicum 3I1B-143 bacteroids. We have performed phase/modulation fluorescence lifetime measurements, as a function of temperature and added quencher KI, in order to resolved the 1.2-ns (blue) and 6.5-ns (red) contributions from the two classes of trp residues. Anisotropy decay studies have also been performed. The binding of NADH dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in fluorescence of bound NADH upon forming a ternary complex with either tartronic acid or D-malate.     

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.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved fluorescence studies of genetically engineered Escherichia coli glutamine synthetase. Effects of ATP on the tryptophan-57 loop.

Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy. The W57L mutant (retains tryptophan at residue 158) and the W158S mutant (retains tryptophan at residue 57) are both characterized by heterogeneous exponential decay kinetics. Global analysis indicates that for the Mn-bound form of the enzyme at pH 7.4 the fluorescence of both tryptophans is best described by a sum of three discrete expontials with recovered lifetimes of 4.77, 1.72, and 0.10 ns for Trp-57 and 5.04, 2.28, and 0.13 ns for Trp-158. The wild-type enzyme also exhibits decay kinetics described by a triple-exponential model with similar lifetime components. The individual tryptophans are distinguishable by the fractional intensities of the resolvable lifetimes. The wild-type and W158S enzymes are dominated by the 5-ns component which provides nearly 60% and 65%, respectively, of the fractional intensity at five wavelengths spanning the emission spectrum. In contrast, the W57L enzyme demonstrates a larger fraction of the 2-ns lifetime species (60%) and only 35% of the longer lifetime component. The substrate ATP induces a shift to approximately 90% of the 5-ns component for the wild-type and W158S enzymes, whereas the W57L protein is essentially unaffected by this ligand. Steady-state quenching studies with iodide indicate that addition of ATP results in a 3.0-3.5-fold decrease in the apparent Stern-Volmer quenching constants for the wild-type and W158S enzymes. Phase/modulation experiments at several iodide concentrations indicate that the median, 2 ns, lifetime component is selectively quenched compared to the 5-ns lifetime component. These results suggest a model where ATP binding results in a shift in the equilibrium distribution of microconformational states populated by Trp-57. ATP shifts this equilibrium nearly completely to the states exhibiting the long-lifetime component which, based on quenching studies, is less solvent-accessible than the conformational states associated with the other lifetime components.     

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.     

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.     

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 yeast phosphoglycerate kinase and its ternary complex

A frequency domain fluorescence study of yeast phosphoglycerate kinase has been performed to observe the effect of substrates on the structure and dynamics of the enzyme. At 20 degrees C and pH 7.2, a biexponential decay is observed for tryptophanyl emission. The short fluorescence lifetime (0.4 ns) component is associated with a spectrum having a 329-nm maximum and a 18.4-kJ/mol activation energy, Ea, for thermal quenching. The long-lifetime (3.5 ns) component has a 338-nm maximum and an Ea of only 7.9 kJ/mol. Tentatively we assign the short and long-lifetime components to Trp-333 and Trp-308. Binding of the substrates ATP and 3-phosphoglycerate leads to a significant increase in the fluorescence lifetime, the red shift of the emission spectrum and in the decrease in the Ea for both components. Acrylamide-quenching studies indicate that the two tryptophan residues have about the same degree of kinetic exposure to the quencher and that the binding of the substrates causes a very slight change in the quenching pattern. These fluorescence studies indicate that the binding of the substrates to phosphoglycerate kinase may influence the conformational dynamics around the two tryptophan residues located on one of the protein's domains.     

Tryptophan fluorescence of terminal deoxynucleotidyl transferase: effects of quenchers on time-resolved emission spectra

Terminal deoxynucleotidyl transferase (EC 2.7.7.31) is a eucaryotic DNA polymerase that does not require a template. The tryptophan environments in calf thymus terminal transferase were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was separated by time-resolved emission spectroscopy. Nanosecond fluorescence decays at 296-nm excitation and various emission wavelengths were deconvolved by global analysis, assuming that the lifetimes but not the relative weighting factors were independent of emission wavelength. The data were fit to three exponentials of lifetimes tau 1 = 1.4 ns, tau 2 = 4.5 ns, and tau 3 = 7.7 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 328, 335, and 345 nm. The accessibility of individual tryptophan environments to polar and nonpolar fluorescence quenchers was examined in steady-state and time-resolved experiments. In the presence of iodide and acrylamide, the steady-state emission spectra shift to the blue. However, at low quencher concentrations, the emission from the 7.7-ns component (maximum 345 nm) is hardly affected, suggesting that this hydrophilic tryptophan environment is buried within the protein. On the other hand, the red shift in the steady-state emission spectrum in the presence of trichloroethanol indicates that the 1.4-ns component (maximum 328 nm) is an exposed hydrophobic tryptophan environment. The results are consistent with an inside-out model for terminal transferase protein, with the more hydrophobic tryptophan(s) near the surface and the most hydrophilic tryptophan(s) in the core.     

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.     

Lifetimes and NADH quenching of tryptophan fluorescence in pig heart cytoplasmic malate dehydrogenase.

The time-resolved and steady state fluorescence properties were measured for pig heart cytoplasmic malate dehydrogenase at pH 6.0 and 8.0. The fluorescence decay can be described by two rate processes, according to the functions: I(t) = 0.7e(-t/1.0) + 0.3e(-t/4.4) for the free enzyme and I(t) = 0.7e(-t/0.8) + 0.3e(-t/2.0 for the enzyme . NADH complex. Quenching by NADH of the tryptophan fluorescence is linear. The only effect of pH is to change the association constant for NADH binding; the fluorescence of the free enzyme and the fluorescence quenching by NADH, I-, and acrylamide are unaffected by pH. Thus there are no changes in conformation of the free enzyme or of the NADH complex over the range of pH 6 to 8.     

Solution conformation of EcoRI restriction endonuclease changes upon binding of cognate DNA and Mg2+ cofactor

EcoRI endonuclease has two tryptophans at positions 104 and 246 on the protein surface. A single tryptophan mutant containing Trp246 and a single cysteine labeling site at the N-terminus was used to determine the position of the N-terminus in the protein structure. The N-termini of EcoRI endonuclease are essential for tight binding and catalysis yet are not resolved in any of the crystal structures. Resonance energy transfer was used to measure the distance from Trp246 donor to IAEDANS or MIANS acceptors at Cys3. The distance is 36 A in apoenzyme, decreasing to 26 A in the DNA complex. Molecular modeling suggests that the N-termini are located at the dimer interface formed by the loops comprising residues 221-232. Protein conformational changes upon binding of cognate DNA and cofactor Mg(2+) were monitored by tryptophan fluorescence of the single tryptophan mutant and wild-type endonuclease. The fluorescence decay of Trp246 is a triple exponential with lifetimes of 7, 3.5, and 0.7 ns. The decay-associated spectra of the 7- and 3.5-ns components have emission maxima at approximately 345 and approximately 338 nm in apoenzyme, which shift to approximately 340 and approximately 348 nm in the DNA complex. The fluorescence quantum yield of the single tryptophan mutant drops 30% in the DNA complex, as compared to 10% for wild-type endonuclease. Fluorescence changes of Trp104 upon binding of DNA were inferred by comparison of the decay-associated spectra of wild type and single tryptophan mutant. Fluorescence changes are related to changes in proximity and orientation of quenching functional groups in the tryptophan microenvironments, as seen in the crystal structures.     

Venom exonuclease. IV. Intrinsic fluorescence of the exonuclease from Crotalus adamanteus venom

The intrinsic fluorescence of the exonuclease isolated from Crotalus adamanteus venom, was studied. The position of its maximum at 335 nm and half-width of the emission band 55 nm (lambda exc. 295 nm) suggested the existence of at least two types of tryptophan residues in the enzyme molecule. Differential analysis of the fluorescence spectra obtained by excitation at 280 and 295 nm revealed about 12.5% contribution of the tyrosine fluorescence in the overall emission excited at 280 nm. The environment of the tryptophan residues in the exonuclease was studied by quenching of their fluorescence with various ionic (NO3-, NO2-, I-, Br- and Cs+) and non-ionic agents (acrylamide, chloroform-methanol). On this basis, fractions of inner (non-polar) and surface tryptophan residues located in charged and neutral regions of the enzyme molecule were evaluated. More than half of the residues (60%) was found in the inner part of the exonuclease while most of its surface tryptophans--in a neutral region(s).     

Long-lived tryptophan fluorescence in phosphoglycerate mutase

Phosphoglycerate mutase (PGM; EC 2.7.5.3) isolated from rat and rabbit muscle has been shown to possess an unusually long-lived fluorescence component when excited by ultraviolet light below 310 nm. On the basis of spectral and physical measurements, this 16.4 (+/- 0.2) ns fluorescence lifetime at room temperature is assigned to a tryptophan residue in an unusual environment. The emission profile of this long-lived tryptophan is red shifted from the other tryptophans of PGM by approximately 25 nm. PGM has been crystallized and sequenced from yeast where it has been shown to be a tetramer with 29K subunits. However, we have not been able to detect the existence of an unusually long-lived fluorescence component in the yeast isomer. The long fluorescence lifetime is lost upon denaturation of rabbit PGM and is partially restored upon introduction of the protein to a nondenaturing environment, suggesting the long lifetime is not the result of a covalent modification. The PGM molecule was studied by a number of techniques including time-resolved tryptophan fluorescence, quenching studies of tryptophan fluorescence, and enzyme activity studies. The long-lived fluorescence has been shown to be statistically quenched by Br-, I-, and Cu2+ in the submillimolar region while the acrylamide quenching shows the tryptophan is marginally accessible to solvent. Characterization of the long-lived fluorescence and its possible sources are discussed.     

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.     

Comparative structural analysis of staphylococcal enterotoxins A and E

Structural analysis of staphylococcal enterotoxins A and E, two functionally and serologically related proteins, has been carried out using circular dichroism, and tryptophan fluorescence quantum yield and quenching. Secondary structures derived from the far-UV circular dichroic spectra revealed that both enterotoxins are in predominantly beta-sheets/beta-turn structures (80-85%). Staphylococcal enterotoxin A has significantly higher alpha-helical content (10.0%) than staphylococcal enterotoxin E (6.5%). Tryptophan fluorescence spectra of both enterotoxins showed maxima at approximately 342 nm, indicating that the fluorescent tryptophan residues are in polar environments. However, the tryptophan fluorescence quantum yields indicated that tryptophan residues are approximately 41% more fluorescent in staphylococcal enterotoxin A than in staphylococcal enterotoxin E. Tryptophan fluorescence quenching by a surface quencher, I-, and a neutral quencher, acrylamide, indicated that at least 1 of the 2 tryptophan residues in both staphylococcal enterotoxins A and E is located on the outer surface of the proteins. This tryptophan residue is in significantly different environments in the two enterotoxins. Six antigenic sites are predicted from the hydrophilicity and secondary structure information; at least four sites are identical. In general, staphylococcal enterotoxins A and E have some structural similarities which are compatible with their common biological activities.