Protein eosine labelling

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... 

I 7 nwsphorescence anisotropy decay In ghosts. The anisotropy decay of eosin-labelled proteins was monitored as a function of time. The data were fitted according to eQuaUon 21. Reivinted in part with permission from Mateyoshi, E. D. and Jovin, T. M. (1991) Biochemistry, 30, 3527-3538. Copyright 1991 American Chemical Society. 

CPI labeled with eosin-SCN in the non activated mode restored photophosphorylation in partially depleted EDTA-chloroplasts and fully depleted NaBr-particles (10). Wheras in the case of the EDTA-particles we could observe very slow rotational motion of the reconstituted eosin-CFI relative to the membrane, in NaBr-part ides we could not detect any rotational motion (up to 500 as) of reconstituted eosin-CFI. We also studied the rotational diffusion of another extrinsic protein of the thylakoid membrane, the ferredoxin-NADP-oxidoreductase (ll), which is probably located in the same stromal region of stacked chloroplasts as CPI and we found very rapid rotation (< 1/is). Only after addition of ferredoxin rotational correlation time decreased to 40 /is. This was interpreted to indicate formation of a ternary complex between ferredoxin-NADP-oxidoreductase ferredoxin and PSI. This revealed rather high lipid fluidy in thylakoids. We tend to assume that the low rotational mobility of the CP0-CF1 complex is caused either by self aggregation or strong interaction with other membrane proteins. 

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