ROOM TEMPERATURE PHOSPHORESCENCE OF AMORPHOUS AGGREGATES AND AMYLOID FIBRILS RESULTING FROM PROTEIN MISFOLDING
V. M. Mazhul,1 E. M. Zaitseva,1 M. M. Shavlovsky,2
O. I. Povarova,3 I. M. Kuznetsova,3 K. K. Turoverov 3, *
1 Institute of Biophysics and Cell Engineering, NASB, Minsk, Belarus,
2 Institute for Experimental Medicine, RAMS, and
3 Institute of Cytology, RAS, St. Petersburg, Russia;
* e-mail: kkt@mail.cytspb.rssi.ru
Using actin, α-lactalbumin and insulin as examples, it was shown that the formation of amorphous aggregates
of proteins and amyloid fibrils leads to an increase in the rigidity of tryprophan and tyrosine residues microenvironment and,
consequently, to the appearance of tryptophan (tyrosine) room temperature phosphorescence (RTP). RTP was used for examining a
slow intramolecular mobility of native (G-, F-form) and inactivated (I) rabbit skeletal muscle actin during the process of GdnHCl
induced protein unfolding. This method made it possible to confirm that an essentially unfolded intermediate precedes the
formation of inactivated actin. It has been found that the kinetic intermediate generated at the early stage of protein
denaturation has no tryptophan RTP, suggesting a high lability of its structure. Symbate changes of integral intensity (relative
quantum yield) and the mean lifetime of RTP during the U* → I transition suggest a gradual increase of the number of
monomers incorporated in the associate (U* → I1 ··· →In → ···
→ I15), which is accompanied by an increase of protein structural rigidity. The rate of inactivated actin
formation (I → I15) is shown to increase with the increase of protein concentration. It is shown that, no matter
what method of inactivation was employed (1-2 M GdnHCl or 3.0-3.5 I urea, Ca2+ removal, incubation at 70 °N, refolding
from completely unfolded state by dialysis from 8 M urea or 6 M GdnHCl), actin transition to the inactivated state is accompanied
by a significant increase in both integral intensity and the mean lifetime of RTP, suggesting the rigid structure of inactivated
actin. It is shown that the lifetime of inactivated actin RTP does not depend on GdnHCl concentration within the limits from 0 to
4 M. On using insulin and α-lactalbumin as examples, it is shown that RTP can be used in studies of fibrillogenesis and
properties of amyloid fibrils.
Key words: room temperature phosphorescence, amyloid fibrils, actin, protein folding, α-lactalbumin,
insulin
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