Time-dependent phosphorescence anisotropy

In Section 2.5 we described the use of time-resolved fluorescence anisotropy for monitoring protein motion on the nanosecond timescale. For motion on much longer timescales, time-resolved phosphorescence anisotropy can be used instead. The latter technique has been employed, for example, to examine the rotational motion of membrane-boimd proteins labelled with the triplet probe eosin (57, 58). Prior to making the measurements, the protein is labelled with eosine-maleimide as described in Protocol 2. 

Eosine labelling of proteins for time-resolved phosphorescence measurements 

Protein buffer (10-100 mM phosphate, Tris or HEPES, pH 7.4), deoxygenated 

Sephadex G-25 column equilibrated with protein bufier, or dialysis tubing 

1 Prepare a 20 mM (—15 mg/ml) stock solution of eosine in DMF just prior to its use. Shield the container from light by wrapping it in aluminium foil. 

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G. B. Strambini and W. C. Galley, Time-dependent phosphorescence anisotropy... 

We have investigated further the rotation of DMPC lipids by incorporating two eosin fatty acid probes (dodecanoyl- and hexadecanoyl-amidoeosin) and measuring the time-dependent phosphorescent anisotropies (37). The eosin moieties of these reporter molecules are located close to the membrane surface. Figure 7 shows typical experimental results at two temperatures. A number of features serve to illustrate the type of information provided by such studies. The phosphorescence emission at both temperatures displayed a time-dependent anisotropy which could be fit to an equation of the form... 

Dependencies of luminescence bands (both fluorescence and phosphorescence), anisotropy of emission, and its lifetime on a frequency of excitation, when fluorescence is excited at the red edge of absorption spectrum. Panel a of Fig. 5 shows the fluorescence spectra at different excitations for the solutes with the 0-0 transitions close to vI vn, and vra frequencies. Spectral location of all shown fluorescence bands is different and stable in time of experiment and during lifetime of fluorescence (panel b)... 

The major reasons for using intrinsic fluorescence and phosphorescence to study conformation are that these spectroscopies are extremely sensitive, they provide many specific parameters to correlate with physical structure, and they cover a wide time range, from picoseconds to seconds, which allows the study of a variety of different processes. The time scale of tyrosine fluorescence extends from picoseconds to a few nanoseconds, which is a good time window to obtain information about rotational diffusion, intermolecular association reactions, and conformational relaxation in the presence and absence of cofactors and substrates. Moreover, the time dependence of the fluorescence intensity and anisotropy decay can be used to test predictions from molecular dynamics.(167) In using tyrosine to study the dynamics of protein structure, it is particularly important that we begin to understand the basis for the anisotropy decay of tyrosine in terms of the potential motions of the phenol ring.(221) For example, the frequency of flips about the C -C bond of tyrosine appears to cover a time range from milliseconds to nanoseconds.(222)... 

Reticulum ATPase [105,106], Owing to the long-lived nature of the triplet state, Eosin derivatives are suitable to study protein dynamics in the microsecond-millisecond range. Rotational correlation times are obtained by monitoring the time-dependent anisotropy of the probe s phosphorescence [107-112] and/or the recovery of the ground state absorption [113— 118] or fluorescence [119-122], The decay of the anisotropy allows determination of the mobility of the protein chain that cover the binding site and the rotational diffusion of the protein, the latter being a function of the size and shape of the protein, the viscosity of the medium, and the temperature. 

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