Fluorescence anisotropy tryptophan residues

The complex anisotropy spectrum of indole was used to determine the absorption spectra corresponding to the 5 — L, and So—i La transitions. We present this example because of its didactic value and its importance for a detailed understanding of the fluorescence from tryptophan residues in proteins (Chapter 16). At any excitation wavelength X, the observed anisotropy is... 

J. R. Lakowicz and G. Weber, Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies, Biophys. J. 32, 591-601 (1980). 

The anisotropy decay of the tryptophan fluorescence of both model peptides and biologically active peptides containing a single tryptophan residue has been determined in various studies. Even in the case of the tripeptide H-Gly-Trp-Gly-OH quenched by acrylamide the anisotropy decay displayed two correlation times with values of 39 and 135 ps. 44 The shorter correlation time was thought to be due to motions of the indole ring relative to the tripeptide. In the case of ACTH(l-24) the fluorescence anisotropy decay of the single tryptophan residue in position 9 of the peptide sequence obtained in phosphate buffer (pH 7, 3.5 °C) was also double-exponential. 29 The shorter rotational correlation time (0 = 92ps)... 

Albani, J.R. (1996). Dynamics of Lens culinaris agglutinin studied by red-edge excitation spectra and anisotropy measurements of 2-p-loluidinylnaphlhalcnc-6-sulfonale (TNS) and of tryptophan residues. Journal of Fluorescence, 6, 199-208. 

Figure 11.4 Steady-state fluorescence anisotropy vs. temperature/viscosity ratio for tryptophan residues of cytochrome b2 core. Data are obtained by thermal variations in the range 10-36°C.

Zargarian, L., Le Tilly, V., Jamin, N., Chaffotte, A., Gabrielsen, O. S., Toma, F. and Alpert, B. 1999, Myb-DNA recognition role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb. Biochemistry, 38, 1921-1929. Zentz, C, Glandieres J. M., El Moshni S. and Alpert, B. 2003, Protein matrix elasticity determined by fluorescence anisotropy of its tryptophan residues. Photochemistry and Photobiology 78, 98-102. 

Besides quantitatively reflecting the binding of a protein to DNA, studies of time-resolved anisotropy provide information about the dynamics of the tryptophan chromophore. In favorable circumstances, the fluorescence emitted from two tryptophan residues in a protein can be re-solved. However, it is desirable that studies be carried out on proteins... 

At present, there is widespread interest in directly measuring the time-dependent processes. This is because considerably more information is av lilable from the time-dependent data. For example, the time-dependent decays of protein fluorescence can occasionally be used to recover the emission spectra of individual tryptophan residues in a protein. The time-resolved anisotropies can reveal the shape of the protein and/or the extent of residue mobility within the protein. The time-resolved energy transfer can reveal not only the distance between the donor and acceptor, but also the distribution of these distances. The acquisition of such detailed information requires high resolution instrumentation and careful data acquisition and analysis. 

In this expression r(/) is the time-dependent anisotropy, 0 the correlation times and g, the fraction of the total anisotropy (r ) which decays with this correlation time. In general we expect one component (tf,) due to rotational diffusion of the protein, and one due to torsional motions of the tryptophan residue, if such motions are significant. In proteins which contain more than a single fluorescent residue there can be energy transfer among the residues, which can appear as a component in the anisotropy decay. The timescale of energy transfer depends upon the distance and orientation between the residues, but there is little information on the timescale of energy transfer between intrinsic fluorophores in proteins. 

Because of their spectral properties, RETcan occur fiom phenylalanine to tyrosine to tryptophan. Also, blue-shifted tryptophan residues can transfra the excitation to longer-wavelength tryptophan residues. In fact, energy transfer has been repeatedly observed in proteins and is one reason for the minor contribution of phenylalanine and tyrosine to the emission of most (Hoteins. The anisotropy displi ed by tyrosine and tryptophan is sensitive to both ov l rotational diffusion of proteins and the extent of segmental motion during the excited-state lifetimes. Hence, the intrinsic fluorescence of proteins can provide considerable information about protein structure and dynamics and is often used to study protein folding and association reactions. In this chapter, we present examples of protein... 

Figure 16.39. Dependence of Ure emission maxima (A), acrylamide quenching constants (B), and steady-state anisotropies (C) of the MLCK pepddes bound to calmodulin on the position of the tryptophan residue. Reprinted, with permission, from O Neil, K. T, Wolfe. H. R., Erickson-Viitanen, S. and DeGrado, W. F., Fluorescence properties of calmodulin-binding peptides reflect alpha-helical periodicity, Science 236 1454-1456, Copyright O 1987, American Association for the Ad-vancement of Stience.

Proteins are generally excited at 295 nm in order to avoid (i) energy transfer from tyrosine to tryptophan residues, which can occur at shorter wavelengths (e.g. at 280 nm where tryptophan has its maximum absorbance) and which depolarizes the fluorescence, and (ii) depolarization of tryptophan fluorescence due to relaxation from its to electronic states. (At shorter wavelengths electrons are excited to both levels, after vdiich electrons in the hi er level relax radiationlessly to the lower, which also depolarizes the fluorescence.) One would like to maximize the initial anisotropy in order to obtain the most sensitivity for polarized fluorescence measurements. 

Another steady-state fluorescence signal that can be measured fairly routinely is the fluorescence anisotropy, r. The anisotropy value for the fluorescence of a tryptophan residue will depend on its rotational fieedom (as expressed by its rotational correlation time, < )) and its fluorescence decay time, t, with immobilized and short-lived excited states having largest r values. Since there is a range of fluorescence lifetime and rotational correlation times for tryptophan residues in native proteins, and since these <() and t values will usually have a narrower range for the unfolded state, it follows that r values will usually change upon unfolding a protein. However, as we have discussed elsewhere (25), r values do not follow equation 1, but instead follow... 

Fig. 11. Variation of the fluorescence properties of a set of tryptophan-containing peptides as a function of the position of the tryptophan in their sequence. The parameter/AVe describes the degree of rigidity and hydrophobicity of the tryptophan s environment it is based on emission maximum, anisotropy, and accessibility to acrylamide. When the values for each of these parameters were similar to those expected for indole in water, a value near 0 was assigned to/, whereas values up to 1.0 were assigned as the fluorescence parameters more closely resembled those observed in very rigid and apolar environments such as the interior of a protein or ethylene glycol at -60°C (Lakowicz, 1983). The values of / calculated for each parameter were then averaged to give /AVe- The dotted curve was generated by fitting a sine wave to the data (period = 3.3 residues). Taken from O Neil et al. (1987).

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