Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state.
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.
Uversky
V N
VN
Institute of Protein Research, Russian Academy of Sciences, Moscow Region, Russia.
Winter
S
S
Löber
G
G
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D012997:Solvents; D013050:Spectrometry, Fluorescence
D000178:Acrylamides; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006146:Guanidines; D011506:Proteins; D012997:Solvents; D020106:Acrylamide; C027132:1-anilino-8-naphthalenesulfonate; D019791:Guanidine