Figure 1.

RBD can only refold reversibly in the presence of disulfides. (a) Schematic of Ox $\to$ Ox and Red $\to$ Ox protocols. RBD is denatured in 5 M GdnHCl in the presence (Red $\to$ Ox) or absence (Ox $\to$ Ox) of 1 mM TCEP to reduce disulfide bonds. Refolding is then initiated via 10-fold dilution into oxidizing buffer containing 5 mM oxidized glutathione. For details, see main text and materials and methods. (b) Intrinsic fluorescence spectra (excitation wavelength of 280 nm) of 1.2 $\mu M$ refolded RBD prepared via the Ox $\to$ Ox (labeled O$\to$O)and Red $\to$ Ox (labeled R$\to$O) protocols, as well as 1.2 $\mu \mathrm{M}$ native protein in 0.5 M GdnHCl to match final denaturant concentration after refolding. Each spectrum represents an average of two independent replicates. (c) Same spectra as in (b) normalized to maximum emission value to emphasize peak shift, alongside normalized spectra of 1.2 $\mu M$ Red $\to$ Ox native sample in which reduction was performed under native conditions (labeled R$\to$O nat.) and sample reduced under native conditions that was not re-oxidized (labeled Red. nat.), both in 0.5 M GdnHCl. All unnormalized spectra are shown in Fig. S1c. (d) bis-ANS fluorescence spectra (excitation wavelength of 385 nm) for all samples in (c). The bis-ANS concentration is $8\mu M$. The colors and line styles match those in (c). (e) bis-ANS fluorescence at 500 nm (385-nm excitation wavelength) as a function of temperature for samples prepared as in (b)–(d). The colors match those in (c).