These results suggest that relatively large environmental changes occur at position 33 during protein unfolding. Previous NMR studies have revealed transitions from the native state to intermediate states of similar structure at 0–0.5 M GdmCl and a subsequent complete transition to an unfolded state at 3.5 M. At 2 M GdmCl, the intermediate states and the unfolded state are present in a 1:1 ratio, whereas only the unfolded state is present at >3.5 M GdmCl. Changes in 1H–15N heteronuclear single-quantum correlation NMR spectra indicate that the loop regions and -strand 3 are the first to be affected at low concentrations (0.5 M), whereas other -strands are affected only at higher concentrations (28). The observed spectral response of 1 at position 33 corresponds well with these findings. In contrast, the fluorescence of mutant protein with 1 at position 16 did not show a large response to unfolding, indicating low changes in local dielectric during structural transitions near this side chain. Removal of copper and zinc ions or reduction of the disulfide bridge resulted in similar unfolding behavior at sites 16 and 33. These studies demonstrate that unnatural amino acid 1 provides a useful probe of structural transitions at defined sites in proteins.
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Fig. 4. Fluorescence spectra of different unfolding states of hSOD containing 1 at position 16 or 33. (A) Fluorescence spectra of hSOD with 1 at position 16 in the presence of different concentrations of GdmCl ( , 0 M; , 0.5 M; , 1.5 M; , 2.0 M; , 3.5 M; , 5.0 M). Samples were measured in 50 mM sodium phosphate (pH 7.2) and 100 mM NaCl containing 3 µM hSOD and 2 eq of ZnCl2 and CuSO4, respectively. Spectra were recorded in 1-nm steps at 22°C by using excitation at 340 nm and a bandpass of 5 nm for both excitation and emission. (B) Fluorescence spectra of hSOD with 1 at position 33 conducted under conditions identical to those in A. (C) Fluorescence intensity of 1 at positions 16 and 33 of hSOD at unfolding states of the protein present at different GdmCl concentrations. The fluorescence intensity of spectra shown in A ( ) and B ( ) at the wavelength where maximal fluorescence is observed ( max) is plotted against the GdmCl concentration. (D) Wavelength of maximal fluorescence intensity ( max) of 1 at positions 16 and 33 of hSOD at unfolding states of the protein present at different GdmCl concentrations. max of spectra shown in A ( ) and B ( ) is plotted against GdmCl concentration. AU, arbitrary units. |
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