Use of Genetically Encoded Dansylalanine as Probe of Protein Unfolding. To demonstrate the utility of dansylamino acid 1 in studies of protein structure and function, it was used as an environmentally sensitive reporter of protein unfolding (13, 17, 18). hSOD has a Greek-key -barrel fold with an external loop and short helical regions at one barrel face near the active site, which contains the copper and zinc ions (27). Loop IV of this region is additionally connected by a disulfide bond (Cys-57 and Cys-146) to -strand 8 of the barrel (Fig. 3). Monomeric hSOD has been shown to undergo reversible and complete unfolding in the concentration range 0–3.5 M guanidinium chloride (GdmCl) (28). To analyze local changes in the barrel core and outer rim regions during unfolding, 1 was incorporated at two different positions, Gln-16 and Trp-33 (Fig. 3). Position 33 lies on the barrel at the center of -strand 3, the first N-terminal strand of Greek-key 1 (27); surface residue Gln-16 forms the N-terminal tip of -strand 2 adjacent to Greek-key -strand 3.
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Fig. 3. Structure of hSOD (Protein Data Bank ID code 1PU0). Protein is colored in green, and Greek-key 1 is shown in red. Amino acids Gln-16 and Trp-33 of -strands 2 and 3 and the disulfide bridge between Cys-57 and Cys-146 are highlighted. Copper and zinc ions at the active site are shown as spheres and are colored in orange and gray, respectively. |
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When 1 was incorporated at positions 16 and 33 in hSOD, the fluorescence intensity increased slightly at both positions (11% and 3% for positions 16 and 33, respectively). The max of 1 also blueshifted compared with the free amino acid ( 14 nm and 27 nm for positions 16 and 33, respectively). These spectral changes likely reflect partial shielding by surface side chains or dielectric differences between bulk water and the protein hydration shell. GdmCl denaturation only weakly affected the emission intensity and wavelength of the Gln-16 1 mutant (Fig. 4 A, C, and D). However, increasing the GdmCl concentration from 0 to 5 M caused a significant redshift in the max (528–544 nm) and decrease in the fluorescence intensity of the Trp-33 1 mutant (Fig 4 B–D). The most striking effects were observed at low GdmCl concentrations (0–1.5 M) followed by moderate changes from 1.5 M to 3.5 M GdmCl and a relatively small change between 3.5 M and 5 M GdmCl (Fig. 4).
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