Local phenomena and distribution of molecular species during the unfolding of heme-free myoglobin in the presence of GdnHCl and urea as seen by time-resolved fluorescence spectroscopy.
We have used time-resolved fluorescence spectroscopy for following the unfolding of apomyoglobin in urea and guanidine hydrochloride (GdnHCl). The data have been compared with those obtained using classical techniques such as CD and steady-state emission spectroscopy. Both the average intensity of the lifetimes and the size of the librational cone of the fluorophores, as measured by time-resolved fluorescence, increased with denaturant concentration and their changes largely preceded the modifications detectable with CD and the shift of the maximum of emission spectra. The data indicate that the changes in the local environments of the tryptophans were completed when the global modification monitored by CD and the emission spectra was still minimal. This suggests that an initial event in the denaturation of apomyoglobin is localized at the tryptophan residues. The correlation times of native apomyoglobin showed the rotational diffusion characteristics of a rigid rotor. In 3.6 M GdnHCl and 7.5 M urea, where the secondary structure is practically absent, the correlation times of the two systems became very short, as expected from the motion of a flexible polymer. In GdnHCl, under conditions of partial unfolding, it was not possible to detect the presence of native totally folded molecular species.
Fronticelli
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University of Maryland, Baltimore, School of Medicine, Department of Biological Chemistry 21201.
Bucci
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Malak
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D001059:Apoproteins / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D009211:Myoglobin / Q000378:metabolism*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014508:Urea / Q000494:pharmacology*
D001059:Apoproteins; D006146:Guanidines; D009211:Myoglobin; C010857:apomyoglobin; D014508:Urea; D019791:Guanidine