Tryptophan fluorescence, energy transfer

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. 

C (57). The fluorescence of either (A) tryptophan or (B) dansyl was measured as a function of time under stopped-flow conditions. Oscilloscope traces of duplicate reactions are shown in each case. Excitation was at 285 nm. Enzyme tryptophan fluorescence was measured by means of band-pass fitter peaking at 360 nm, and dansyl emission was measurea by a 430-nm cutoff filter. Scale sensitivities for (A) and (B) are 50 and 500 mV/div, respectively. The existence of the ES complex is signaled by either (A) the suppression of enzyme tryptophan fluorescence (quenching by the dansyl group) or (Ib) enhancement of the substrate dansyl group fluorescence (energy transfer from enzyme tryptophan). 

Lysine and Tyr side chains can quench tryptophan fluorescence by transferring a proton to the excited indole ring [59, 75]. The phenolate side chain of ionized tyrosine also can quench by resonance energy transfer and possibly by transferring an electron to the indole [51]. 

Zauner, G. Lonardi, E. Bubacco, L. Aartsma, T. J. Canters, G. W Tepper, A. W. J. W. Tryptophan-to-dye fluorescence energy transfer applied to oxygen sensing by using type-3 copper proteins. Chem.-Eur. J. 2007,13, 7085-7090. 

The intramolecular distances measured at room temperature with the AEDANS FITC pair were similar in the Ca2Ei and E2V states [297]. Ca and lanthanides are expected to stabilize the Ej conformation of the Ca -ATPase, since they induce a similar crystal form of Ca -ATPase [119,157] and have similar effects on the tryptophan fluorescence [151] and on the trypsin sensitivity of Ca -ATPase [119,120]. It is also likely that the vanadate-stabilized E2V state is similar to the p2 P state stabilized by Pi [418]. Therefore the absence of significant difference in the resonance energy transfer distances between the two states implies that the structural differences between the two conformations at sites recorded by currently available probes, fall within the considerable error of resonance energy transfer measurements. Even if these distances would vary by as much as 5 A the difference between the two conformations could not be established reliably. 

Liao F, Xie Y, Yang X et al (2009) Homogeneous noncompetitive assay of protein via Forster-resonance-energy-transfer with tryptophan residue(s) as intrinsic donor(s) and fluorescent ligand as acceptor. Biosens Bioelectron 25 112-117... 

Visser NV, Borst JW, Hink MA, van Hoek A, Visser AJWG (2005) Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins. Biophys Chem 116 207-212... 

Weber G. (1960) Fluorescence Polarization Spectrum and Electronic Energy Transfer in Tyrosine, Tryptophan and Related Compounds, Biochem. J. 75, 335-345. 

Eisinger(55) also noted that it is difficult to obtain accurate data with phenylalanine as the donor and either tryptophan or tyrosine as the acceptor. The source of this problem is the weak S, - S0 absorption of phenylalanine compared to that of tyrosine or tryptophan, which leads to considerable experimental uncertainty in measuring the sensitized acceptor emission. This error may account for the finding of Kupryszewska et al.<56> that the sensitization of the acceptor fluorescence was less than the quenching of the donor fluorescence in their study of phenylalanine-to-tyrosine energy transfer... 

As the investigation of the interactions between H DAC inhibitors and the enzymes are an important issue, competition assay systems are helpful implements in facilitating the characterization of inhibitor binding. Such a competition binding assay that has been developed for histone deacetylases is based on fluorescence resonance energy transfer (FRET) between tryptophan residues of the histone deacetylase and a fluorescent HDAC inhibitor [38]. In competition with other... 

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. 

With site-directed mutation and femtosecond-resolved fluorescence methods, we have used tryptophan as an excellent local molecular reporter for studies of a series of ultrafast protein dynamics, which include intraprotein electron transfer [64-68] and energy transfer [61, 69], as well as protein hydration dynamics [70-74]. As an optical probe, all these ultrafast measurements require no potential quenching of excited-state tryptophan by neighboring protein residues or peptide bonds on the picosecond time scale. However, it is known that tryptophan fluorescence is readily quenched by various amino acid residues [75] and peptide bonds [76-78]. Intraprotein electron transfer from excited indole moiety to nearby electrophilic residue(s) was proposed to be the quenching... 

In a multitryptophan protein, the fluorescence quantum yield of tryptophans can be additive or not. In the first case, there is no interference between Trp residues. In the second case, energy transfer between tryptophan residues can influence the quantum yield of each of them. 

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. 

Tyrosine is more fluorescent than tryptophan in solution, but when present in proteins, its fluorescence is weaker. This can be explained by the fact that the protein tertiary structure inhibits tryosine fluorescence. Also, energy transfer from tyrosines to tryptophan residues occurs in proteins inducing a total or important quenching of tyrosine fluorescence. This tyrosine — tryptophan energy transfer can be evidenced by nitration of tyrosine residues with tetranitromethane (TNM), a highly potent pulmonary carcinogen. Because TNM specifically nitrates tyrosine residues on proteins, the effects of TNM on the phosphorylation and dephosphorylation of tyrosine, and the subsequent effects on cell proliferation, can be investigated. 

The tryptophan fluorescence intensity of native CGTase is eightfold higher than that of a L-tryptophan solution with respect to the total amount of tryptophan. This suggests that CGTase supports energy transfer from tyrosyl to tryptophan residue(s). 

The fluorescence intensity of CGTase tryptophan residues decreases (Figure 7.13) after treatment of the enzyme with increasing concentrations of TNM and purification of the modified enzyme on co-polymer. Tryptophan fluorescence intensity of the 8 mM-TNM-modified CGTase (0.03 /uM 1 tryptophan) is similar to that of free L-tryptophan (0.034 /xM 1). The loss of tryptophan fluorescence observed during nitration may then be related to elimination of this energy transfer. 

CGTase nitration induces an 11.5 nm shift in the fluorescence maximum to shorter wavelengths (A.max = 326.5 nm instead of 338 nm for 8 mM TNM), suggesting a relative increase in the buried tryptophan fluorescence. Tyr -> Trp energy transfer may then involve solvent-exposed residue(s). 

---