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.     

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.     

Physicochemical characterization of bovine retinal arrestin

The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mM-1, cm-1 using the tyrosinate-tyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford colorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60 degrees C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed.     

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.     

The ultraviolet fluorescence spectra of DPN-linked isocitrate dehydrogenase from bovine heart.

The emission maximum of DPN-linked isocitrate dehydrogenase from bovine heart shifted from 316 nm to 324 nm as the excitation wavelength was varied from 265 nm to 300 nm. This shift was accompanied by a nonproportional change in fluorescence intensity. Comparisons of the emission spectra of model compounds in aqueous buffer at pH 7.07 and n-butanol showed that lowered solvent polarity led to a blue shift of the peak of free tryptophan without significant change of fluorescence intensity, whereas the fluorescence intensity of tyrosine amide increased markedly without change in emission maximum. The emission peak of mixtures of tryptophan and tyrosine amide shifted to shorter wavelengths as the proportion of tyrosine amide increased. The results suggest a major contribution of tyrosine to the overall fluorescence of the dehydrogenase. DPNH caused quenching and a blue shift of the protein fluorescence maximum when excited between 270 nm and 290 nm, indicating that the two tryptophan residues per subunit of enzyme are located in different microenvironments of the protein and that DPNH may interact preferentially with the residue emitting at the longer wavelength.     

Fluorescence and circular-dichroism properties of pig intestinal calcium-binding protein (Mr=9000), a protein with a single tyrosine residue.

Spectral properties of pig intestinal Ca2+-binding protein (CaBP) and its apoprotein have been examined by fluorescence, absorption and c.d. Direct fluorescence from some of the five phenylalanine residues is observed and excitation spectra show that there is also energy transfer from some phenylalanine residues to the tyrosine. Absorption and c.d. spectra show that the tyrosine hydroxy group does not ionize significantly below pH 12. Tyrosine fluorescence is reversibly quenched by a lysine residue with a pK of 10.05 in the Ca2+ form. At low pH the tyrosine fluorescence is enhanced with transitions with pK values of approx. 4.2. The c.d. spectrum of the Ca2+ form shows a decrease of the ellipticity band at 276nm with a transition similar to that of the fluorescence titration. The apoprotein, however, shows an additional transition with a pK of about 6. The results are interpreted in terms of the recently published structure of the cow intestinal CaBP [Szebenyi, Obendorf & Moffat (1981) Nature (London) 294, 327-332]. The single tyrosine has a very high pK, although it apparently lies on the surface of the protein molecule.     

The role of the single tryptophan residue in the structure and function of ribonuclease T1

The previously reported method for the preparation of Kyn 59-RNase T1 and NFK 59-RNase T1 has been improved, and these two proteins have been obtained in high purity. Kyn 59-RNase T1, fully active for the hydrolysis of GpA and GpC, emitted a 35-fold-enhanced fluorescence of kynurenine relative to acetylnurenine amide with an emission maximum at 455 nm upon excitation at 380 nm. The polarity of the environment of Kyn 59 estimated from the emission maximum corresponded to a dielectric constant of 6. Upon excitation at 325 nm, NFK 59-RNase T1, less active than Kyn 59-RNase T1, exhibited a quenched N'-formylkynurenine fluorescence with an emission maximum at 423 nm, from which the value of 12 was obtained as the dielectric constant of the surroundings of residue 59. In both modified proteins, distinct tyrosine fluorescence appeared on excitation at 280 nm. The detection of an energy transfer from tyrosine to residue 59 suggests that the tertiary structure is very similar in Kyn 59-RNase T1 and native RNase T1. With guanidine hydrochloride, Kyn 59-RNase T1 was less stable than the native protein. Carboxymethylation at Glu 58 was shown to stabilize the active site of the modified enzyme. Based on the information collected for Kyn 59-RNase T1, the local environment and possible roles of the sole tryptophan residue in RNase T1 are discussed.     

Aromatic interactions in homeodomains contribute to the low quantum yield of a conserved, buried tryptophan

Trp 48, a conserved, buried residue commonly found in the hydrophobic core of homeodomains, has an unusually low fluorescence quantum yield. Chemical denaturation of Drosophila homeodomains Engrailed and Antennapedia(C39S) result in a four-fold increase in quantum yield, while unfolding of Ultrabithorax causes a twenty-fold enhancement. Global analysis of time-resolved fluorescence decay monitored at multiple emission wavelengths reveals sub-nanosecond lifetime components which dominate the overall intensity. Based on structure and sequence analysis of several homeodomains, we deduce that quenching is due to a transient, excited-state NH ellipsis pi hydrogen bond involving Trp 48 and a conserved aromatic residue at position 8. Additionally, both time-resolved fluorescence of indole-benzene mixtures and an electrostatic model of the proposed tryptophan-aromatic interaction substantiate different aspects of this mechanism. A survey of the Protein Data Bank reveals many proteins with tryptophan-aromatic pairs where the indole nitrogen participates in a NH ellipsis pi hydrogen bond with the ring of another aromatic residue. Chemical denaturation of one protein found in this survey, human fibronectin type III module 10, causes an enhancement of the fluorescence quantum yield. This unique interaction has implications for many other systems and may be useful for studying larger, multi-tryptophan containing proteins.     

Importance of tyrosine for structure and function of mitochondrial malate dehydrogenases

Mitochondrial malate dehydrogenase from pig and chicken both contain one tyrosine/subunit with highly red-shifted spectrum, most probably involved in a hydrogen bond with a carboxylate group. The spectral changes of this tyrosine can be used as an indicator for alkaline denaturation, acid transition and coenzyme binding. Acid transition is coupled with breaking of this bond by protonation as monitored by loss of absorbance at 290 nm. Activity is lost and fluorescence intensity is increased at slightly higher pH, thus indicating increased mobility of the indicator and most probably of the whole protein prior to protonation of the indicator-tyrosine.     

The S100-b Protein: Tyrosine Residues Do Not Exhibit an Abnormal Fluorescence Spectrum

The β subunit of the bovine brain S100-b protein (ββ) lacks tryptophyl residue but contains one tyrosine. Our experiments show that this protein is characterized by a typical tyrosine fluorescence spectrum, with a maximum at 303 nm. Identical fluroescence properties were found for the rat brain S100-b protein. Comparison with the fluorescence spectrum of the bovine brain S100-a’ protein (α'β), which contains a tryptophan residue in the α’ subunit, enables us to demonstrate that the recent report describing an abnormal fluorescence spectrum for the bovine brain S100-b protein may result from a contamination of the S100-b by the S100-α’ protein.     

Fluorimetric and spectrophotometric studies of DPN-linked isocitrate dehydrogenase from bovine heart. Properties of tyrosyl and tryptophyl residues.

The emission maximum of DPN-linked isocitrate dehydrogenase in pH 7.07 buffer is shifted from 317 to 324 nm and fluorescence intensity is decreased when the excitation wave-length is varied from 270 to 290 nm; in 0.2 M KOH, where the fluorescence of tyrosyl residues is almost completely quenched, a further substantial decline in quantum yield of protein fluorescence and a red shift of the emission peak to 339 nm occur. The latter should be due mainly to tryptophyl residues. The enzyme contains 9.4 tyrosyl residues per subunit of molecular weight 42,000 determined spectrophotometrically (295 nm) at pH 13, in good agreement with a tyrosine content of 9.7 by amino acid analysis. No more than 1.1 tyrosyl residues per subunit can be detected up to pH 10.6 at 7 degrees upon prolonged incubation. The increase in absorption at 295 nm with increasing pH is related to loss of enzyme activity and results in a red shift of the emission maximum, and decreased fluorescence intensity. Treatment of the enzyme in a Li+-containing buffer at pH 7.5 with an excess of N-acetylimidazole results in (a) modification of 1.1 tyrosyl residues per subunit, (b) a 30% decrease in enzyme activity, (c) a 6-nm red shift in emission maximum, and (d) a decrease in fluorescence intensity. Manganous DL-isocitrate (1.06 mM) prevents the acetylation of the enzyme. Deacetylation of the O-acetylated enzyme by hydroxylamine completely restores the enzyme activity and reverses the spectral changes. The acetylation studies indicate that the reactive tyrosyl residue does not participate directly in catalysis but may be involved in maintaining the proper conformation of the active enzyme center. A net of 1 of the 2 tryptophyl residues per subunit is perturbed immediately by a number of solvents. This perturbation is not affected by manganous isocitrate, whereas exposure of tyrosyl residues occurs only with time and is prevented by the substrate. The perturbation of the tryptophyl residue is accompanied by a red shift of the fluorescence emission maximum. The more exposed tryptophyl residue may contribute to the energy transfer from protein to nucleotides since the quenching of protein fluorescence upon binding of DPN+, DPNH, or ADP by enzyme results in a blue shift of the emission maximum. Manganous DL-isocitrate (1.06 mM) quenches protein fluorescence by 16% without a shift in emission peak and does not affect the relative extent of fluorescence quenching induced by the nucleotides.     

Spectroscopy of intermolecular interactions of a tyrosine chromophore. III. Classification of the state of tyrosine residues in proteins based on their electron spectra

Absorption and fluorescence spectra of some tyrosine-containing proteins were analysed. Comparison of the peculiarities of fluorescence and absorption of the tyrosine chromophore in the model compounds and proteins suggested a new classification of the states of tyrosine residues in proteins: I -- tyrosyls with hydrated OH-group (lambda mf approximately equal to 304 nm); II -- tyrosyls, whose hydroxyl group forms the hydrogen bond inside the protein in a hydrophobic surrounding or in the globular fold in structured water layer (lambda mf = 306-307 nm); III -- tyrosyls whose OH-group is deprotonated in the excited state (lambda mf approximately equal to 330-350 nm).     

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

mPlum is a far‐red fluorescent protein with emission maximum at ～650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far‐red emission. Ultrafast time‐resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum‐E16Q and ‐I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far‐red emission. Both the far‐red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.     

Association of the internal membrane protein with the lipid bilayer in influenza virus. A study with the fluorescent probe 12-(9-anthroyl)-stearic acid

The lipid fluorescent probe 12-(9-anthroyl)-stearic acid was introduced into the lipid bilayer of influenza virus particles. Fluorescent energy transfer was observed from the viral protein to the probe. This transfer persisted after removal of the glycoprotein spikes which cover the outside of the viral particle, demonstrating that the energy donor was an internal protein. It was concluded that the energy donor was the non-glycosylated membrane protein (M protein), the major protein component of the spikeless particle. Analysis of the emission spectrum of the spikeless particle excited at 275 nm shows that a substantial portion of the fluorescence arises from tyrosine residues, in contrast to most other proteins which contain both tryptophan and tyrosine.It is suggested that the donor residue(s) are located no more than 11 Å exterior to the bilayer surface, and that a portion of the M protein may penetrate into the bilayer.     

The intrinsic tyrosine fluorescence of histone H1. Steady state and fluorescence decay studies reveal heterogeneous emission

In wavelength-resolved steady state spectra we observe three different kinds of emission from histone H1, a class A protein with only a single tyrosine residue. Unfolded H1 emissions that peak at approximately 300 and 340 nm can both be excited maximally at approximately 280 nm. Another, peaking much further to the red at approximately 400 nm, can be excited maximally at approximately 320 nm. The 300-nm fluorescence can be resolved by lifetime measurements into three components with decay times of approximately 1, 2, and 4 ns. On sodium-chloride-induced refolding of H1, simplification of the emission properties occurs. The 340 and 400-nm components disappear while the two shorter lifetime components of the 300-nm band diminish in amplitude and are replaced by the 4-ns decay. We believe that the 340-nm emission is tyrosinate fluorescence resulting from excited-state proton transfer. The origin of the 400-nm emission remains uncertain. We assign the 1 and 2-ns components of the 300-nm emission to two states of tyrosine in denatured H1 and the 4-ns decay to fluorescence of the single tyrosine residue in the globular region of refolded H1. Our results support the contention that salt induced folding of H1 is a cooperative two state process, and permit us to better understand the previously reported increases in fluorescence intensity and anisotropy on salt-induced folding.     

Fluorescent properties of b-type ferredoxins]

Fluroescent spectra of six b-type ferredoxins of plant and animal origins were obtained. All investigated proteins do not contain tryptophan. The emission maxima of the native proteins, apoproteins prepared by various methods, and denaturated proteins are compared. The effects of pH, ionic strength and ferricyanide on the ferredoxins fluorescence were studied. "Unusual" emission at 340nm noted previously for adrenal ferredoxin was observed for spinach and Chenopodium album ferredoxins too. The localization of tyrosine fluorescent maximum at 340nm in the ferredoxins is not due to interaction of tyrosine with the iron-sulfur center. The data obtained allow to suggest that the tyrosine residues in ferredoxins have different environments.     

Interaction of tyrosine 65 of RecA protein with the first and second DNA strands

We investigated the structure of the active RecA-DNA complex by analyzing the environment of tyrosine residue 65, which is on the DNA-binding surface of the protein. We prepared a modified RecA protein in which the tyrosine residue was replaced by tryptophan, a natural fluorescent reporter, and measured the change in its fluorescence upon binding of DNA and cofactor. The fluorescence of the inserted tryptophan 65 (Trp65) was centered at 345 nm, indicating a partly exposed residue. Binding cofactor, adenosine 5'-O-3-thiotriphosphate (ATPgammaS), alone at a low salt concentration did not change the fluorescence of Trp65, confirming that the residue is not close to the nucleotide. In contrast, the binding of single-stranded DNA quenched the fluorescence of Trp65 in both the presence and absence of ATPgammaS. Trp65 fluorescence was also quenched upon binding a second DNA strand. The fluorescence change depended upon the presence and absence of ATPgammaS, reflecting the difference in the DNA binding. These results indicate that residue 65 is close to both the first and second DNA strands. The degree of quenching depended upon the base composition of DNA, suggesting that the residue 65 interacts with the DNA bases. Binding of DNA with ATPgammaS as well as binding of ATPgammaS alone at high salt concentration shifted the fluorescence emission peak from 345 to 330 nm, indicating a change from a polar to a non-polar environment. Therefore, the environment change around residue 65 would also be linked to a change in conformation and thus the activation of the protein.     

Conformational changes of disulfide isomerase C induced by guanidine hydrochloride

Unfolding--refolding of Escherichia coli disulfide isomerase C (DsbC) induced by GdnHCl was studied by intrinsic fluorescence. Interpretation of experimental fluorescence data was done together with the analysis of protein 3D structure. It is shown that although Cys 141 is the next neighbour of a single tryptophan residue Trp 140, sulfur atoms of the disulfide bond Cys 141--Cys 163 are far apart from the indole ring and cannot quench its fluorescence, while the potential quenchers are Met 136 and His 170. It has been revealed that, though each subunit of DsbC contains eight tyrosine residues, only three tyrosine residues (Tyr 171, Tyr 38 and Tyr 52) contribute to the bulk fluorescence of the molecule. The character of intrinsic fluorescence intensity changes induced by GdnHCl (equilibrium and kinetic data), the character of parametric dependencies between fluorescence intensity recorded at 320 and 365 nm, and the existence of an isosbestic point of protein fluorescence spectra in solutions with different GdnHCl concentrations, allowed suggesting a one-step character of DsbC denaturation. The reversibility of this process is also shown.     

Luminescent pyrimidine hydrazide oligomers with peptide affinity

The modular synthesis of pyrimidine oligohydrazides and their peptide binding ability are reported. Ethylene glycol substituents ensure water solubility of the compounds. The pattern of hydrogen bond donors and hydrogen bond acceptors resembles the functionalities of a peptide backbone, and intramolecular hydrogen bonds restrict conformational mobility. The pyrimidine heterocycles show emission at 423 nm if either excited with light of 320 nm or by a FRET process from a nearby Trp residue. This property is useful for the luminescent detection of interactions with peptides and proteins.     

Solution-state characteristics of the ultraviolet A-induced visible fluorescence from proteins

In response to illumination by ultraviolet-A (UV-A) light, proteins in solid form are now known to display a visible blue fluorescence, ostensibly on account of excitation transitions of loosely-held electrons within peptide bond orbitals engaged in hydrogen bonding. Because the CO and NH atom groups in peptide bonds are generally engaged in extensive hydrogen bonding in globular proteins even in aqueous solution, one could argue that proteins in solution must also display this novel blue fluorescence. Here, using high concentrations to enhance detectability, two globular proteins, γ-crystallin, and lysozyme, are shown to fluoresce visibly, exhibiting: (a) two excitation maxima, at ∼315 nm and ∼385 nm, (b) maximal emission at 425 nm in 100 mg/ml lysozyme and 465 nm in 100 mg/ml γ-crystallin, (c) a time-resolved emission decay that is best fitted by a sum of three exponentials with lifetimes of 3.14, 0.46, and 9.08 ns, respectively, and comparable relative amplitudes of around 30--40 percent each, and (d) a weak CD spectrum displaying a positive band at ∼385 nm and a negative band at ∼465 nm. While the wavelength of maximal emission (^emλ_max) in lysozyme is the same for all protein concentrations, the ^emλ_max of γ-crystallin varies with protein concentration, suggesting a certain degree of conformation dependence.