Tryptophan, protein emission spectrum

Additional evidence for conformational changes in the transporter has come from measurement of the intrinsic fluorescence of the protein tryptophan residues, of which there are six, in the presence of substrates and inhibitors of transport. The fluorescence emission spectrum of the transporter has a maximum at about 336 nm, indicating the presence of tryptophan residues in both non-polar environments (which would emit maximally at about 330 nm) and in polar environments (which would emit at 340-350 nm) [154], The extent of quenching by the hydrophilic quencher KI indicates that more than 75% of the fluorescence is not available for quenching, and so probably stems from tryptophan residues buried within the hydrophobic interior of the protein or lipid bilayer [155]. Fluorescence is quenched... 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

Plastocyanin from parsley, a copper protein of the chloroplast involved in electron transport during photosynthesis, has been reported to have a fluorescence emission maximum at 315 nm on excitation at 275 nm at pH 7 6 (2°8) gjncc the protein does not contain tryptophan, but does have three tyrosines, and since the maximum wavelength shifts back to 304 nm on lowering the pH to below 2, the fluorescence was attributed to the emission of the phenolate anion in a low-polarity environment. From this, one would have to assume that all three tyrosines are ionized. A closer examination of the reported emission spectrum, however, indicates that two emission bands seem to be present. If a difference emission spectrum is estimated (spectrum at neutral pH minus that at pH 2 in Figure 5 of Ref. 207), a tyrosinate-like emission should be obtained. 

Figure 3.2 shows the fluorescence and phosphorescence emission spectrum from tobacco mosaic virus coat protein. These spectra are fairly typical of the tryptophan emission spectra observed from proteins at room temperature. 

Differences between the spectra of fluorescence and phosphorescence are immediately obvious. For all tryptophans in proteins the phosphorescence spectrum, even at room temperature, is structured, while the fluorescence emission is not. (Even at low temperatures the fluorescence emission spectrum is usually not structured. The notable exceptions include a-amylase and aldolase, 26 protease, azurin 27,28 and ribonuclease 7, staphylococcal endonuclease, elastase, tobacco mosaic virus coat protein, and Drosophila alcohol dehydrogenase 12. )... 

If the absorbance spectrum of a small molecule overlaps the emission spectrum of tryptophan and if the distance between them is small, quenching will be observed. If binding of such a molecule to a protein quenches tryptophan fluorescence, tryptophan must be in or near (<2 nm away from) the binding site. 

Figure 8.6 clearly indicates that in the presence of free tryptophan in solution, there is no binding of TNS on the amino acid. However, in the presence of bovine serum albumin, TNS shows a fluorescence emission spectrum, indicating that TNS is bound to the protein. 

Figure 3.18. (a) Kibbon diagram of acyl-CoA binding protein ACBP (Kraulis, P. J. 1991. J. Appl. Cryst. 24, 946>950), based on the MMR structure coordinates of Andersen and Poulsen (Andersen, K V, and Poulsen, F. M , 1993, J. Biomol. MMR 3,271-284.) The two tryptophan residues and the mutated C-terminal isoleucine are shown In ball and stick, (b) Absortance spectrum (solid line) and fluorescence emission spectrum with excitation at 280 nm (dashed line) of iS pM ACBP.I86C labeled with IAEDANS(in 20 mMMa-acetate, pH 5.3). 

Human immunoefficiency virus (HIV) is the agent responsible for the acquired immunodeficiency syndrom (AIDS). HIV-1 protease is among the targets identified for chemotherapy. It is a homodimeric aspartyl protease of 99 amino acid residues per monomer. The protein contains two Trp residues (W6 and W42) per monomer located in different environment. The fluorescence emission spectrum of the protein shows a peak at 341 nm 215 nm) indicating that the Trp residues are responsible for the protein emission. Also, tlie position of this peak is blue-shifted compared to the fluorescence maximum of Tryptophan in water and thus the average exposure of the Trp residues to the solvent is not total. Trp residues are partly buried within the hydrophobic core of the protein. 

A very common method to put into evidence the fluorescence spectrum of each component is the decay associated spectra. This method allows combining the dynamic time-resolved fluorescence data with the steady-state emission spectrum. The fluorescence decays are globally or individually fitted to the n-exponential function (n being the number of species, tryptophan or tyrosine residues for example), and the decay associated spectra are constructed (Krishna and Periasamy, 1997). Figure 8.27 displays the decay associated spectra of Trp residues of ai-acid glycoprotein performed by Hof et al (1996). The authors found four fluorescence lifetimes instead of the three we obtained. They attributed the peak of 337 nm to Trp-122 with a location between the surface of the protein and its core. The determination of the degree of hydrophobicity around the Trp residues showed that Trp-25 residue is located in a hydrophobic environment and Trp-160 is the most exposed to the solvent. 

The Log-normal analysis method shows also that binding of calcofluor at high concentrations disrupts the protein structure. This method is very sensitive to the modifications occurring around the fluorophore. In fact, in presence of folded protein structure, the calculated emission spectrum fits perfectly the experimental one (Fig. 8.33). However, when the structure of the protein is altered, we obtain a calculated spectrum that is different from the experimental one (Fig. 8.34, spectrum b, max 337 nm). Also, in this case, we can derive two other emission spectra that correspond to the hydrophobic (kmax 330 nm) and surface Trp residues ( max 355 nm). This deconvolution is not possible in the presence of a compact protein (Fig. 8.33). Therefore, the Log-normal analysis allows comparing disrupted and compact proteins with two or three classes of Trp residues, since in the case of a compact protein the analysis gives a result identical to that obtained for a free tryptophan in solution. 

The effects of solvent polarity are best imderstood by specific examples. To model the fluorescence emission of proteins we examine spectra for iV-acetyl-L-tryptophanamide (NATA). This molecule is analogous to tryptophan in proteins. It is a neutral molecule, and its emission is more homogeneous than that of tryptophan itself. In solvents of increasing polarity the emission spectra shift towards longer wavelengths (Fig. 5). The emission maxima of NATA in dioxane, ethanol and water are 333, 344 and 357, respectively. These solvents are non-viscous, so the emission is dominantly from the relaxed state (Fig. 4). The spectral shifts can be used to calculate the change in dipole moment which occurs upon excitation [6]. More typically, the emission spectrum for a sample is compared with that foimd for the same fluorophore in various solvents, and the environment judged as polar or non-polar. While this approach is qualitative, it is simple and reliable, and does not involve the use of theoretical models or complex calculations. 

One example selective quenching is provided by apoazurin, that is, an azuiinlackii copper. Apoazurin Ade has two tryptophan residues, one surface readue and cme buried residue. The emission spectrum df diis protein, alc g with those d its single-tryptc dian rdsUives. was shown in Figure 16.16. For our present purpc es, the prc ierties d the pro in with ot without ( k>) are... 

An obvious application of time-resolved proton fluorescence is to resolve die contributions of individual tryptophan residues in multi-tiyptophan proteins. This is accomplished Ity measuring die intensity decay across die emission spectrum of die protein, resulting in wavelengdi-dependent data. 

Previously, we have studied fluorescence quenching in unsolvated dye-derivatized biomolecules by performing measurements of fluorescence lifetime, intensity and emission spectrum as a function of temperature. These studies have identified that fluorescence quenching is dependent on conformational dynamics which lead to interactions between the dye and a Trp (Tryptophan) side chain in peptides [34,35] and small protein [36,37] structures. 

The indole group of tryptophan (Trp) has a higher molar extinction coefficient than the phenolic and phenyl side chains of tyrosine (Tyr) and phenylalanine (Phe), respectively (see Table 1, Chapter 12). Thus although its quantum yield is similar to that of lyr, its fluorescence emission is much mote intense. Furthermore, its excitation spectrum overlaps the emission spectrum of Tyr and therefore fluorescence resonance energy transfer (FRET, see Chapters 2 and 3) from lyr to Trp occurs readily when both residues are in close proximity (i.e. located in the same protein molecule) and favourably orientated. The intrinsic fluorescence of a protein is therefore dominated by the contribution from Trp... 

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