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Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule stateстатья
- Авторы: Uversky Vladimir N., Winter Stefan, Löber Günter
- Журнал: Biophysical Chemistry
- Том: 60
- Номер: 3
- Год издания: 1996
- Издательство: Elsevier BV
- Местоположение издательства: Netherlands
- Первая страница: 79
- Последняя страница: 88
- DOI: 10.1016/0301-4622(96)00009-9
- Аннотация: The conformational transitions starting with the native protein, passing the molten globule state and finally approaching the unfolded state of proteins was investigated for bovine carbonic anhydrase B (BCAB) and human alpha-lactalbumin (alpha-HLA) by means of fluorescence decay time measurements of the dye 8-anilinonaphthalene-1-sulphonic acid (8-ANS). Stepwise denaturation was realized by using the denaturant guanidinium chloride (GdmCl). It was shown that 8-ANS bound with protein yields a double-exponential fluorescence decay, where both decay times considerably exceed the decay time of free 8-ANS in water. This finding reflects the hydrophobic environment of the dye molecules attached to the proteins. The fluorescence lifetime of the short-time component is affected by protein association and can be effectively quenched by acrylamide, indicating that 8-ANS molecules preferentially bind at the protein surface. The fluorescence lifetime of the long-time component is independent of the protein and acrylamide concentration and may be related to protein-embedded dye molecules. Changes of the long lifetime component upon GdmCl-induced denaturation and unfolding of BCAB and alpha-HLA correlate well with overall changes of the protein conformation. The transition from native protein to the molten globule state is accompanied by an increase of the number of protein-embedded 8-ANS molecules, while the number of dye molecules located at the protein surface decreases. For the transition from the molten globule to the unfolded state was the opposite behaviour observed.
- Добавил в систему: Уверский Владимир Николаевич