Tryptophan fluorescence lifetimes

Table 22 Tryptophan fluorescence lifetimes and relative emission intensities in aqueous solution in function of pH citric acid-sodium phosphate buffer. Ajk 280 nm emission through 350 nm cut-offfilter, T = 20 °C...

Table 23 Tryptophan fluorescence lifetimes and relative emission intensities in...

Tryptophan fluorescence lifetime values and relative emission int ... 

Malo GD, Pouwels LJ, Wang M, Weichsel A, Montfort WR, Rizzo MA, Piston DW, Wachter RM (2007) X-ray structure of Cerulean GFP a tryptophan-based chromophore useful for fluorescence lifetime imaging. Biochemistry 46 9865-9873... 

RNA. Both proteins contain no tryptophan S8 contains three tyrosines, and S15 contains two tyrosines. The tyrosine emission of these two proteins represents a case in which the quantum yield is higher in the native than the denatured protein. The average fluorescence lifetime, however, was little affected by denaturation no change was observed for S8, and the average lifetime decreased about 12% for S15. The collisional quenchers 1 and Cs + had essentially equivalent access to the tyrosines of both proteins, either in the native or denatured state, and comparison of the bimolecular quenching constants with that of free tyrosine suggested that the tyrosines were all well exposed. The mechanism for the reduction in quantum yield upon denaturation, apparently a static interaction, has not been elucidated. 

One would expect that lowering the temperature or increasing the viscosity of the solvent would increase the width of the lifetime distribution, since both factors may affect the rate of transitions between microstates. If this rate is high as compared with the mean value of the fluorescence lifetime, the distribution should be very narrow, as for tryptophan in solution. When the rate of transitions between microstates is low, a wide distribution would be expected. 

The range of six orders of magnitude for lifetimes of tryptophan phosphorescence in proteins at room temperature is larger than for fluorescence. The lower limit for fluorescence lifetime is about 0.5 ns, while the upper limit is 8 ns.(21> Typical values range from 3 to 5 ns. 

Room temperature phosphorescence can be observed from dried proteins. Sheep wool keratin(47) has a phosphorescence lifetime of 1.4 s. Six lyophilized proteins were shown to exhibit phosphorescence at room temperature.(48) The spectra were diffuse, and the lifetime was non-single-exponential, which the authors interpreted as due to inhomogeneous distribution of tryptophans. As the protein was hydrated, the phosphorescence lifetime decreased. This decrease occurred over the same range of hydration where the tryptophan fluorescence becomes depolarized. Hence, these results are consistent with the idea that rigidity of the site contributes to the lifetimes. 

INTRINSIC AND EXTRINSIC FLUORESCENCE. Intrinsic fluorescence refers to the fluorescence of the macromolecule itself, and in the case of proteins this typically involves emission from tyrosinyl and tryptopha-nyl residues, with the latter dominating if excitation is carried out at 280 nm. The distance for tyrosine-to-tryp-tophan resonance energy transfer is approximately 14 A, suggesting that this mode of tyrosine fluorescence quenching should occur efficiently in most proteins. Moreover, tyrosine fluorescence is quenched whenever nearby bases (such as carboxylate anions) accept the phenolic proton of tyrosine during the excited state lifetime. To examine tryptophan fluorescence only, one typically excites at 295 nm, where tyrosine weakly absorbs. [Note While the phenolate ion of tyrosine absorbs around 293 nm, its high pXa of 10-11 in proteins typically renders its concentration too low to be of practical concern.] The tryptophan emission is maximal at 340-350 nm, depending on the local environment around this intrinsic fluorophore. 

A study by Petrich et al. (1987) on the fluorescence lifetimes of excited tryptophans in azurin has proved exceptionally interesting, especially in light of the studies to be reviewed below on ascorbate oxidase. By comparing the lifetimes of tryptophan fluorescence of three azurins— Az-Pae (only one tryptophan, Trp-48), A.faecalis [Az-Afe (one tryptophan, Trp-118)] and A. denitrificans [Az-Ade (two tryptophans, Trp-48), and Trp-118)] in both holo and apo forms—the authors found that (1) there is virtually no fluorescence quenching in the apo forms (2) the decay of... 

A number of studies on the fluorescence decay of tyrosine, tyrosine derivatives, and small tyrosyl peptides have been carried out. 36-38 Whereas the tyrosine zwitterion and tyrosine derivatives with an ionized a-carboxy group exhibited monoexponential fluorescence decay (x = 3.26-3.76 ns), double- or triple-exponential decay was observed in most other cases. As in the case of the tryptophan model compounds, the complex decay kinetics were again interpreted in terms of rotamer populations resulting from rotation around the C —Cp bond. There is evidence to indicate that the shorter fluorescence lifetimes may arise from rotamers in which the phenol ring is in close contact with a hydrated carbonyl group 36 37 and that a charge-transfer mechanism may be implicated in this quenching process. 39 ... 

Table 7.1 shows the fluorescence lifetime, quantum yield, and position of the emission maximum of tryptophan in basic, neutral, and acidic solvents. One can see that the three parameters are not the same in the three media. The different types of protonation explain this variation. 

Also, upon binding of a ligand to a protein, Trp observables (intensity, polarization, and lifetime) can be altered, and so one can follow this binding with Trp fluorescence. In proteins, tryptophan fluorescence dominates. Zero or weak tyrosine and phenylalanine fluorescence results from energy transfer to tryptophan and/or neighboring amino acids. 

Albani, J.R. (2007). New insights in the interpretation of tryptophan fluorescence. Origin of the fluorescence lifetime and characterization of a new fluorescence parameter in proteins the emission to excitation ratio. /. of fluorescence, in press. 

Figure 10.8 Quenching of the tryptophan fluorescence of or -acid glycoprotein (4 /.(Ml by TNS at20°C. Intensity (a) and lifetime (b) variation. Reproduced from Albani, J.R., Sillen, A., Engelborghs, Y. and Gervais, M. (1999). Photochemistry and Photobiology, 69,22-26, with permission of the American Society for Photobiology.

The cytochrome b2 core from the yeast Hansenula anomala has a molecular mass of 14 kDa, and its sequence shows the presence of two tryptophan residues. Their fluorescence intensity decay can be adequately described by a sum of three exponentials. Lifetimes obtained from the fitting are equal to 0.054,0.529, and 2.042 ns, with fractional intensities equal to 0.922, 0.068, and 0.010. The mean fluorescence lifetime, r0, is 0.0473 ns. 

Figure 14.8 shows the plot of Trp to heme distance obtained by fluorescence energy transfer as a function of tc2 (from 0 to 4) for the two shortest fluorescence lifetimes (solid curve). The five dotted lines in the figure show the crystallographic distances between tryptophan residues and heme. The intersection between the crystallographic lines and the energy-transfer plots is equal to the tc2 corresponding to the specific tryptophan. 

This explanation for the origin of the non-exponential decays of tryptophan fluorescence, however, cannot rationalize the results of time-resolved fluorescence anisotropy in these proteins. Beec-hem and Brand review the time-resolved fluorescence of tryptophans in proteins (32). The following conclusions concerning time-resolved fluorescence can be derived i) fluorescence decays with at least two-lifetime components, when internal rotation of tryptophan is observed from the time-resolved fluorescence anisotropy, ii) fluorescence decays... 

Phase fluorometers utilize continuous irradiation by a beam of lighf thaf is sinusoidally modulated. If the frequency of fhe modulation is sef correcfly, there will be a phase difference in the modulation of the fluorescent emission that will depend upon x. Phase fluorometry can yield the same information as does pulse fluorometry.327432,133 gy ysing two or more modulation frequencies the decay rates and fluorescence lifetimes for various chromophores in a protein can be observed. For example, the protein colicin A (Box 8-D) contains three tryptophans W86, W130, and W140. Their fluorescence decays with lifetimes Xj, Xy X3 of -0.6-0.9 ns, 2.0-2.2 ns, and 4.2-4.9 ns at pH 7. While X3 originates mainly from W140, both of the other tryptophans contribute to x and X2. Changes in fluorescence intensify with pH reflect a pfC value of... 

The large environmental dependence of the ll-(3-hexyl-l-indolyl)undecyltri-methylammonium bromide fluorescence lifetime and fluorescence spectrum has led to its continued use as a fluorescence probe for micellar systems.76 Fluorescence-detected circular dichroism, a technique which appears to have considerable potential, has been applied to the determination of the c.d. spectra of L-tryptophan and L-cystine and two macromolecular systems.78... 

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