Tryptophan anisotropy

Strambini and Galley have used tryptophan anisotropy to measure the rotation of proteins in glassy solvents as a function of temperature. They found that the anisotropy of tryptophan phosphorescence reflected the size of globular proteins in glycerol buffer in the temperature range -90 to -70°C.(84 85) Tryptophan phosphorescence of erythrocyte ghosts depolarized discontinuously as a function of temperature. These authors interpreted the complex temperature dependence to indicate protein-protein interactions in the membrane. 

Fig. 2. The tryptophan anisotropy of the Ada protein ( ) on interaction with DNA at two protein concentrations (a) 2.0 M (b) 0.2 M. No change was observed in the steady-state fluorescence signal (O). The dashed lines are extrapolations to estimate the site size. The solid line is the theoretical curve for an association constant of 4 x lo A/" and a site size of 7 bp assuming no site-site interactions. (From Takahashi et a(. )...

The measurement of the tryptophan anisotropy has the additional advantage that it can be used to establish the polymeric state of the protein. In the case of the Ada protein, the anisotropy was independent of protein concentration, consistent with the observation that the protein remains in a monomeric state over a wide range of protein concentrations. An increase in the steady-state tryptophan anisotropy of the trp repressor on binding a 26 bp operator sequence has also been observed. 

A. Intrinsic Tryptophan Anisotropy Decay of Liver Alcohol Dehydrogenase... 

There are several biologically important peptides which contain tyrosine but not tryptophan. These include small molecules with molecular weights of about 1000 or less. Molecules such as oxytocin, vasopressin, and tyrocidine A are cyclic, while others such as angiotensin II and enkephalin are linear. Schiller 19) has reviewed the literature up through 1984 on fluorescence of these and several other peptides. One major finding that has been reported recently is that the anisotropy and fluorescence intensity decays of many peptides are complex. This is especially evident in some of the tyrosine-containing peptides, and we expect that there will be considerable effort made over the next few years toward understanding the physical basis for these complex kinetics. 

The elucidation of the intramolecular dynamics of tryptophan residues became possible due to anisotropy studies with nanosecond time resolution. Two approaches have been taken direct observation of the anisotropy kinetics on the nanosecond time scale using time-resolved(28) or frequency-domain fluorometry, and studies of steady-state anisotropy for xFvarying within wide ranges (lifetime-resolved anisotropy). The latter approach involves the application of collisional quenchers, oxygen(29,71) or acrylamide.(30) The shortening of xF by the quencher decreases the mean time available for rotations of aromatic groups prior to emission. 

In order to avoid complications caused by excitation energy transfer between tryptophan residues, most investigations have been performed with proteins containing one tryptophan residue per molecule. When studying protein solutions, there are difficulties in separating the effects of rotation of entire protein molecules and of the chromophores themselves relative to their environment in the protein matrix. It is usually assumed that intramolecular motions are more rapid and manifest themselves as short-lived components of anisotropy decay curves or in depolarization at short emission lifetimes. 

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 value of A in the absence of motion is referred to as the limiting anisotropy, A0. This value is related to the relative direction of the absorption and emission dipoles. For tryptophan, /40is negative, which indicates that the absorption and emission dipoles are approximately perpendicular to each other.(83)... 

Berger and Vanderkooi(88) studied the depolarization of tryptophan from tobacco mosaic virus. The major subunit of the coat protein contains three tryptophans. The phosphorescence decay is non-single-exponential. At 22°C the lifetime of the long component decays with a time constant of 22 ms, and at 3°C the lifetime is 61 ms. The anisotropy decay is clearly not singleexponential and was consistent with the known geometry of the virus. 

The long lifetime of phosphorescence allows it to be used for processes which are slow—on the millisecond to microsecond time scale. Among these processes are the turnover time of enzymes and diffusion of large aggregates or smaller proteins in a restricted environment, such as, for example, proteins in membranes. Phosphorescence anisotropy is one method to study these processes, giving information on rotational diffusion. Quenching by external molecules is another potentially powerful method in this case it can lead to information on tryptophan location and the structural dynamics of the protein. 

H. Kirn and W. C. Galley, Rotational mobility associated with the protein moiety of human serum lipoproteins from tryptophan phosphorescence anisotropy measurements, Can. J. Biochem. Cell Biol. 61, 46-53 (1983). 

If a collisional quencher of the fluorophore is also incorporated into the membrane, the lifetime will be shortened. The time resolution of the fluorescence anisotropy decay is then increased,(63) providing the collisional quenching itself does not alter the anisotropy decay. If the latter condition does not hold, this will be indicated by an inability to simultaneously fit the data measured at several different quencher concentrations to a single anisotropy decay process. This method has so far been applied to the case of tryptophans in proteins(63) but could potentially be extended to lipid-bound fluorophores in membranes. If the quencher distribution in the membrane differed from that of the fluorophore, it would also be possible to extract information on selected populations of fluorophores possibly locating in different membrane environments. 

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)... 

Fig. 10. Highly schematic representation of the orientation of several tryptophan-containing peptides with respect to calmodulin. (A) With tryptophan in position 1, the indole is located on the hydrophilic side of the helix and is exposed to solvent. Peptides with tryptophan on this face of the helix should exhibit emission maxima near that of indole in water ( 350 nm), a small anisotropy, and a high accessibility for acrylamide quenching. (B) In position 2, the tryptophan is partially exposed at the interface between the peptide and calmodulin. Peptides with a tryptophan in this location should have fluorescence properties that are intermediate between example A and C. (C) The tryptophan is on the hydrophobic side of the helix and is almost entirely buried. The emission maximum should be strongly blue-shifted, the anisotropy should be large, and the accessibility to acrylamide quenching low. Taken from O Neil et al. (1987).

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).

Figure 4. Ultrafast hydration correlation function c(t) of tryptophan. The c(t) can be fit with a stretched biexponential model as shown. The inset shows the fluorescence anisotropy dynamics of tryptophan after ultrafast ultraviolet (UV) absorption. The internal conversion between La and Lb states occurs within 80 fs. The free rotation time of tryptophan in bulk water is 46 ps.

Figure 19 shows the typical fluorescence transients of TBE from more than 10 gated emission wavelengths from the blue to the red side. At the blue side of the emission maximum, all transients obtained from four Trp-probes in the cubic phase aqueous channels drastically slow down compared with that of tryptophan in bulk water. The transients show significant solvation dynamics that cover three orders of magnitude on time scales from sub-picosecond to a hundred picoseconds. These solvation dynamics can be represented by three distinct decay components The first component occurs in about one picosecond, the second decays in tens of picoseconds, and the third takes a hundred picoseconds. The constmcted hydration correlation functions are shown in Fig. 20a with anisotropy dynamics in Fig. 20b. Surprisingly, three similar time scales (0.56-1.431 ps, 9.2-15 ps, and 108-140 ps) are obtained for all four Trp-probes, but their relative amplitudes systematically change with the probe positions in the channel. Thus, for the four Trp-probes studied here, we observed a correlation between their local hydrophobicity and the relative contributions of the first and third components from Trp, melittin, TME to TBE, the first components have contributions of 40%, 35%, 26%, and 17%, and the third components vary from 32%, to 38%, 43%, and 53%, respectively. The...

The inset shows the correlation functions in the short time range. The hydration correlation function of tryptophan in bulk water is also shown (dashed line) for comparison, (b) fs-resolved fluorescence anisotropy dynamics of Trp probes in the aqueous channels of the cubic phase. For clarity, the anisotropy of TME is not shown. The relaxation of Trp in bulk water (46 ps) is also shown (dashed line) for comparison. 

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.

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