Triplet state of tryptophan in proteins. 2. Differentiation between tryptophan residues 62 and 108 in lysozyme.

We have used optically detected magnetic resonance (ODMR) to characterize the degree of solvent availability of the tryptophan residues in lysozyme that are likely to be responsible for the observed phosphorescence. From the phosphorescence spectra, ODMR zero-field splittings (zfs), and ODMR line widths, we concur with the X-ray structure [Blake, C. C., Mair, G. A., North, A. C. T., Phillips, D. C., & Sarma, V. R. (1967) Proc. R. Soc. London, ser. B 167, 365-377] that Trp-62 behaves as an exposed residue and Trp-108 is buried. In addition, we present evidence that ODMR can be used in conjunction with conventional phosphorescence to evaluate the degree of order in the microenvironments of tryptophan in a protein containing several tryptophans. By the specific modification of residues Trp-62 and Trp-108, we have identified those portions of the ODMR lines in the native enzyme that are due to those specific residues. Barring major enzyme conformational changes in the vicinity of unmodified tryptophan residues when Trp-62 or Trp-108 are selectively modified, we find that Trp-108 dominates both the phosphorescence and the ODMR signals in native lysozyme. The results are discussed in view of previous fluorescence findings.     

Investigation of the membrane-active peptides melittin and glucagon by photochemically induced dynamic-nuclear-polarization (photo-CIDNP) NMR

The photochemically induced dynamic-nuclear-polarization (photo-CIDNP) NMR technique was used to investigate the membrane-active peptides melittin and glucagon. The experiments were performed both in the absence and presence of phospholipid vesicles in order to study the topography of the membrane-bound state. From the results it can be concluded that the melittin peptide chain is oriented in such a way that the single tryptophan residue (Trp19) reaches into the membrane. In the case of glucagon, a binding interaction with vesicle membranes is indicated within the pH range 2-10, whereby the single tryptophan residue (Trp25) is buried in the lipid bilayer and the tyrosine and histidine residues are exposed to the aqueous solvent.     

Conformational heterogeneity of the copper binding site in azurin. A time-resolved fluorescence study.

Comparison of the fluorescence spectra and the effect of temperature on the quantum yields of fluorescence of Azurin (from Pseudomonas fluorescens ATCC-13525-2) and 3-methylindole (in methylcyclohexane solution) provides substantive evidence that the tryptophan residue in azurin is completely inaccessible to solvent molecules. The quantum yields of azurin (CuII), azurin (CuI), and apoazurin (lambda ex = 291 nm) were 0.052, 0.054, and 0.31, respectively. Other evidence indicates that there is no energy transfer from tyrosine to tryptophan in any of these proteins. The fluorescence decay behavior of each of the azurin samples was found to be invariant with emission wavelength. The fluorescences of azurin (CuII) and azurin (CuI) decay with dual exponential kinetics (tau 1 = 4.80 ns, tau 2 = 0.18 ns) while that of apoazurin obeys single exponential decay kinetics (tau = 4.90). The ratio of pre-exponentials of azurin (CuII), alpha 1/alpha 2, is found to be 0.25, and this ratio increases to 0.36 on reduction to azurin (CuI). The results are interpreted as originating from different interactions of the tryptophan with two conformers of the copper-ligand complex in azurin.     

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins

We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade). Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments. From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale. The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale. The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II). The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor. The involvement of protein matrix and donor mobility for electron transfer is discussed. The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood. The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics.     

Conformational changes in azurin from Pseudomona aeruginosa induced through chemical and physical protocols

Azurin from Pseudomona aeruginosa is a small copper protein with a single tryptophan (Trp) buried in the structure. The Gibbs free energies associated with the folding of holo azurin, calculated monitoring Trp fluorescence and changes in absorbance on the ligand-to-metal band, are different because these techniques probe their local environments, thereby being able to probe different conformational changes. The presence of an intermediate state was observed during the chemical denaturation of the protein. Upon denaturation, a 30-fold increase is observed in the magnitude of the quenching constant of the tryptophan fluorescence by acrylamide, because this residue becomes more accessible to the quencher. Entrapping the protein in sol-gel materials lowers its stability possibly because the solvation properties of the macromolecule are changed. The thermal denaturation of azurin immobilized in a sol-gel monolith is irreversible, which tends to rule out an aggregation mechanism to account for the irreversibility of the denaturation of the protein free in solution. Unlike the Cu(II) ion, the Gd(III) ion accommodates in site B of azurin with high affinity and the folding free energy of Gd-azurin is larger than that of apo azurin.     

Optically detected magnetic resonance of tryptophan residues in Escherichia coli ssb gene product and E. coli plasmid-encoded single-stranded DNA-binding proteins and their complexes with poly(deoxythymidylic) acid

Optically detected magnetic resonance (ODMR) spectroscopy has been applied to several single-stranded DNA-binding (SSB) proteins encoded by conjugative plasmids of enteric bacteria. Fluorimetric equilibrium binding isotherms confirm their preferential binding to single-stranded DNA and polynucleotides and reveal a limited protein solubility at low ionic strength. The plasmid SSB-like proteins show the highest affinity for polydeoxythymidylic acid; these complexes are the least sensitive to disruption by salt. ODMR data on these complexes suggest the existence of stacking interactions between tryptophan residue(s) and thymine bases, as evidenced by spectral red shifts of the tryptophan phosphorescence 0,0 band, reduction of the magnitude of D zero field splitting parameter, and a dramatic reversal of the polarity of the ODMR signals. Wavelength-selected ODMR results point to the existence of two distinct tryptophan sites in these complexes. The triplet state properties of the red-shifted site are drastically altered by its interaction with the thymine bases. The chromosomal Escherichia coli SSB protein-poly(dT) complex shows an additional tryptophan site with zero field splitting parameters similar to those of the free protein. This site can be attributed to Trp-135, which is missing in each of the other plasmid SSB proteins, suggesting that this particular residue is not involved in the interaction with polynucleotides.     

Dipole–dipole interactions between tryptophan side chains and hydration water molecules dominate the observed dynamic stokes shift of lysozyme

The fluorescence intensity of tryptophan residues in hen egg-white lysozyme was measured up to 500 ps after the excitation by irradiation pulses at 290 nm. From the time-dependent variation of fluorescence intensity in a wavelength range of 320–370 nm, the energy relaxation in the dynamic Stokes shift was reconstructed as the temporal variation in wavenumber of the estimated fluorescence maximum. The relaxation was approximated by two exponential curves with decay constants of 1.2 and 26.7 ps. To interpret the relaxation, a molecular dynamics simulation of 75 ns was conducted for lysozyme immersed in a water box. From the simulation, the energy relaxation in the electrostatic interactions of each tryptophan residue was evaluated by using a scheme derived from the linear response theory. Dipole–dipole interactions between each of the Trp62 and Trp123 residues and hydration water molecules displayed an energy relaxation similar to that experimentally observed regarding time constants and magnitudes. The side chains of these residues were partly or fully exposed to the solvent. In addition, by inspecting the variation in dipole moments of the hydration water molecules around lysozyme, it was suggested that the observed relaxation could be attributed to the orientational relaxation of hydration water molecules participating in the hydrogen-bond network formed around each of the two tryptophan residues.     

Investigation of the role of individual tryptophan residues in the binding of Escherichia coli single-stranded DNA binding protein to single-stranded polynucleotides. A study by optical detection of magnetic resonance and site-selected mutagenesis

Fluorescence and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been employed to study the complexes formed between single-stranded polynucleotides and Escherichia coli ssb gene products (SSB) in which tryptophans 40, 54, and 88 are selectively, one residue at a time, replaced by phenylalanine using site-specific oligonucleotide mutagenesis. Fluorescence titrations and ODMR results indicate that tryptophans 40 and 54 are the only tryptophan residues in E. coli single-stranded DNA binding protein that are involved in stabilizing the protein-nucleic acid complexes via stacking interactions. Wavelength-selected ODMR measurements on E. coli SSB reveal the presence of two spectrally distinct tryptophan sites (Khamis, M. I., Casas-Finet, J. R., and Maki, A. H. (1987) J. Biol. Chem. 262, 1725-1733). Our present results indicate that tryptophan 54 belongs to the blue-shifted site, while tryptophan 40 belongs to the red-shifted site of the protein.     

Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence.

Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue. Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water. In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein. The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin. Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue. However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue. The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid. Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein.     

Photooxidative changes of lysozyme with 337.1 nm laser radiation

Summary Initial photoinduced oxidative changes in the protein lysozyme were studied using the 337.1 nm radiation from a pulsed nitrogen laser without exogenous sensitizing compounds. Irradiation of lysozyme and tryptophan in aerated solution results in the temperature and solvent dependent loss of tryptophan absorption and fluorescence, and the appearance of fluorescent daughter products, primarily N-formyl-kynurenine and kynurenine. Exposures that resulted in 15% loss of tryptophan fluorescence produced no measurable loss in enzymatic activity. Fluorescence quenching experiments on irradiated lysozyme at low conversion percentage suggest that an exposed residue (Trp-62) is favored as an initial target of attack.     

Characterization of the tryptophan residues of Escherechia coli alkaline phosphatase by phosphorescence and optically detected magnetic resonance spectroscopy.

The phosphorescence and zero field optically detected magnetic resonance (ODMR) of the tryptophan (Trp) residues of alkaline phosphatase from Escherechia coli are examined. Each Trp is resolved optically and identified with the aid of the W220Y mutant and the terbium complex of the apoenzyme. Trp(109), known from earlier work to be the source of room-temperature phosphorescence (RTP), emits a highly resolved low-temperature phosphorescence (LTP) spectrum and has the narrowest ODMR bands observed thus far from any protein site, revealing a uniquely homogeneous local environment. The decay kinetics of Trp(109) at 1.2 K reveals that the major triplet population (70%) undergoes inefficient crystallike spin-lattice relaxation by direct interaction with lattice phonons, the remainder being relaxed efficiently by local disorder modes. The latter population is smaller than is typical for protein sites, suggesting an unusual degree of local rigidity and order consistent with the long-lived RTP. Trp(220) emits a broader LTP spectrum originating to the blue of Trp(109). It has typically broad ODMR bands consistent with local heterogeneity. The LTP of Trp(268) has an ill-defined origin blue shifted relative to Trp(220) and ODMR frequencies consistent with a greater degree of solvent exposure. Trp(268) has noticeable dispersion of its decay kinetics, consistent with quenching at the triplet level by a nearby disulfide residue.     

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.     

Study of reactivity of tryptophan residues in serum albumins and lysozyme by N-bromosuccinamide fluorescence quenching

The kinetics of quenching by N-bromosuccinamide of the fluorescence of tryptophan residues in various proteins and peptides were studied using the stopped-flow technique. Human serum albumin, which has a single tryptophan residue located in a hydrophobic fold, showed biphasic kinetics; one component was second order and the other first order, the rate for the latter component being independent of the concentration of quencher. Bovine serum albumin, which has in addition a tryptophan residue at the surface of the molecule, showed triphasic kinetics; two components corresponded to those for human serum albumin, and there was a faster second-order component resulting from the surface tryptophan. It is concluded that reaction with the buried tryptophan involves the initial second-order formation of a complex in which the quencher is located at the mouth of a hydrophobic fold, and that this is followed by a first-order conformational change which allows interaction to occur between the quencher and the tryptophan. The kinetics of lysozyme fluorescence quenching at pH 5.4 showed two relaxations whose rates were proportional to the N-bromosuccinamide concentration. The results of kinetic and titration experiments suggest that a molecule of lysozyme contains at least two groups of tryptophan residues of significantly different reactivities. The faster component probably reflects the bromination of the tryptophan residues at the active site. An octapeptide consisting of residues 22 to 29 of glucagon showed essentially the same quenching kinetics as glucagon itself, and Leu-Trp-Met showed the same behavior as Gly-Trp-Gly. The results indicate that quenching by N-bromosuccinamide provides a useful indication of the accessibility of tryptophan residues, and that side reactions do not significantly affect the kinetics.     

葡萄糖淀粉酶色氨酸残基及底物结合位点的研究

 本文用N-溴代琥珀酰亚胺(NBS)对葡萄糖淀粉酶进行特异性修饰,当酶分子表面有3个色氨酸残基被修饰后,酶活力完全丧失。用邹氏图解法测得酶活性中心有一个色氨酸残基是必需的。如果在酶液中加入不同的底物再用NBS氧化,用荧光发射和荧光猝灭光谱检测表明,底物对酶分子有不同程度的保护作用。在被测试的三种底物中,这种保护能力依为糊精>淀粉>麦芽糖。     

Hydrogen-deuterium exchange of the tryptophan residues in bovine α-lactalbumin

Effects of deuteration on the Raman spectrum of a tryptophan residue have been examined. The 1386 cm^?1 line of deuterated tryptophan residue has been found to be useful for tracing the hydrogen-deuterium exchange reaction of this residue in a protein. An examination on bovine α-lactalbumin at pH 6.4 and at 20°C indicates that two of the four tryptophan residues exchange with a rate constant much greater than 9 × 10^?4 sec^?1, while the other two exchange with a rate constant of 4 × 10^?5 sec^?1. The latter two have been assigned to Trp 28 and Trp 108 of this protein. The kinetics of hydrogen-deuterium exchange reaction of completely “free” tryptophan residue have been examined by a proton magnetic resonance study on tryptophan itself. By taking the result of this examination into account, the chance of exposure to the solvent for Trp 28 or Trp 108 has been estimated to be 3 × 10^?6 at pH 6.4 and at 20°C.     

The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties

Special analysis of the tryptophan residue localization in the structure of the macromolecule of Pseudomonas aeruginosa azurin made it possible to prove many explanations in the existing literature of the extraordinary fluorescence properties of this protein, to choose between various contradictory conclusions and in some cases even to make new interpretations of the known experimental data. It has been revealed that the microenvironment of the tryptophan residue is in principle formed by non-polar hydrocarbon groups. The density of the microenvironment is not very high and there are cavities around the ring. The conformation of the side chain of the tryptophan residue is unstrained. These results have been analysed in connection with available data on the unique short-wave fluorescence spectrum position and the existence of the high-frequency indole ring mobility with significant amplitude. Judging by the distance between tryptophan and tyrosine residues and their mutual orientation, the conclusion was made that there is no energy transfer from Tyr 72 to tryptophan and that the efficiency of the energy transfer from Tyr 108 to tryptophan is about 0.5. The mechanism of the dramatic increase in fluorescence efficiency when the copper atom is removed has been discussed with due regard to the fact that the 'blue' copper centre is displaced from the indole ring by more than 10 A.     

Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens.

A strain of Pseudomonas fluorescens contains an azurin with no tryptophan and two tyrosines. This protein is interesting because it allows one to study both the structure of azurin and the emission of tyrosines in proteins. Comprehensive measurements were carried out including spectrophotometric and fluorimetric titration, fluorescence quantum yield, fluorescence polarization, and I- quenching. In the copper-containing protein, almost independent of the copper ion oxidation, the fluorescence quantum yield is approximately 60% of that of the apoprotein. The latter has the remarkable property that its quantum yield is even greater than free tyrosine. The two tyrosines in the metalloprotein have different pKa's, 10.75 and 12.78, but there is only one average pKa, 10.9 in the apoprotein. The polarization of the fluorescence at 310 nm (290-nm excitation) is 0.32 for the metalloproteins and 0.34 for the apoprotein. I- hardly quenches the fluorescence. The conclusion is that the two tyrosines are inaccesible to the solvent, located in nonpolar environments, larger than or equal to 20 A apart, and not adjacent to the disulfide bridge.     

Probing alpha-helical secondary structure at a specific site in model peptides via restriction of tryptophan side-chain rotamer conformation.

The relationship between alpha-helical secondary structure and the fluorescence properties of an intrinsic tryptophan residue were investigated. A monomeric alpha-helix forming peptide and a dimeric coiled-coil forming peptide containing a central tryptophan residue were synthesized. The fluorescence parameters of the tryptophan residue were determined for these model systems at a range of fractional alpha-helical contents. The steady-state emission maximum was independent of the fractional alpha-helical content. A minimum of three exponential decay times was required to fully describe the time-resolved fluorescence data. Changes were observed in the decay times and more significantly, in their relative contributions that could be correlated with alpha-helix content. The results were also shown to be consistent with a model in which the decay times were independent of both alpha-helix content and emission wavelength. In this model the relative contributions of the decay time components were directly proportional to the alpha-helix content. Data were also analyzed according to a continuous distribution of exponential decay time model, employing global analysis techniques. The recovered distributions had "widths" that were both poorly defined and independent of peptide conformation. We propose that the three decay times are associated with the three ground-state chi 1 rotamers of the tryptophan residue and that the changes in the relative contributions of the decay times are the result of conformational constraints, imposed by the alpha-helical main-chain, on the chi 1 rotamer populations.     

Stacking interactions of tryptophan residues and nucleotide bases in complexes formed between Escherichia coli single-stranded DNA binding protein and heavy atom-modified poly(uridylic) acid. A study by optically detected magnetic resonance spectroscopy

The complexes formed between Escherichia coli single-stranded DNA binding protein (SSBP) and the heavy atom-modified single-stranded polynucleotides poly(5-BrU) and poly(5-HgU) are investigated using optically detected magnetic resonance (ODMR) methods. In these complexes the triplet state properties of the tryptophan residues are subjected to the external heavy atom effect generated by bromine and mercury atoms and are characterized by a shortened triplet state lifetime and the appearance of the otherwise dark [D] + [E] slow passage ODMR signal. These features provide direct evidence for close range interactions between tryptophan residue(s) and the nucleotide bases in the complexes. The extent of the triplet state lifetime reduction in the case of the SSBP-poly(5-HgU) complex together with steric considerations of the complex structure is consistent only with a van der Waals contact between the perturbed molecule and the heavy atom perturber by means of a stacking interaction. Fast passage ODMR measurements show a lifetime for a sublevel of the perturbed tryptophan chromophore(s) in this complex on the order of 1 ms. The amplitude-modulated phosphorescence microwave double resonance technique captures selectively the broadened and red-shifted phosphorescence spectrum of the heavy atom-perturbed tryptophan residue(s). This work supports a model for the binding of SSBP to single-stranded polynucleotides in which the bases are inserted into hydrophobic regions of the protein, where they are likely to undergo stacking interactions with the indole moiety of buried tryptophan residues.     

Difference in association of Tl(I) with gramicidin A and gramicidin B in trifluoroethanol determined by Tl-205 NMR

The thallium-205 chemical shift was determined as a function of temperature for the thallium(I) complexes of gramicidin A and gramicidin B in 2,2,2-trifluoroethanol. From the difference in magnitude of the induced chemical shift it was determined that gramicidin B does not bind the Tl(I) ion as well as does gramicidin A. This result may explain the lower single-channel conductance of gramicidin B relative to gramicidin A. Cabon-13 NMR studies strongly indicate that the binding site for gramicidin A and B is at teh tryptophan end of the molecule and that replacement of tryptophan residue at position 11 in gramicidin A with a phenylalanine to form gramicidin B produces a significant structural change at the tryptophan end of the molecule, but has little effect on the N-terminus.