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Figure 5
Steady-state fluorescence spectroscopy on RepA–WT and its fragments. (A) Fluorescence emission spectra of W94 ( excitation = 295 nm) in RepA proteins (8.32  M) at 20°C. Spectra (identified as a–h in the inset and plotted to the same scale in arbitrary fluorescence units) were acquired in ammonium sulfate buffer (folded state), or in the same buffer supplemented with 4.5 M GuHCl (unfolded state; only that for RepA–WT is plotted since the others are similar). The spectrum for the buffer including GuHCl is also shown (the Raman band for water is the small peak at  330 nm). (B) Maximum emission wavelength for each spectrum in (A). The ratios between the fluorescence intensities for the native and unfolded states (Fbuff/FGuHCl) are also shown. Data are compatible with all fragments having W94, to a different extent, buried in the protein hydrophobic core (an indication of being folded). (C) Extrinsic fluorescence emission of bis-ANS (16.64 M) after incubation with 8.32 M of each RepA protein (for 15 min at 20°C). The inset shows the identity of each sample, including equimolar mixtures of fragments that mimic full length and  N37 RepA. The emission intensity at 500 nm ranks the degree of exposure of hydrophobic regions and the compactness of RepA domains.
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