Figure 2.

Reduced RBD folding landscape intrinsically favors a highly-aggregation-prone nonnative intermediate. (a) Size exclusion chromatography UV absorbance traces, normalized to maximum value, for native RBD and refolded RBD per Red $\to$ Ox protocol, defined as in Fig. 1. Dashed vertical lines indicate elution positions for standards with indicated molecular weights. (b) Two possible scenarios that may explain observed misfolding and aggregation. Solid curves with three minima represent energy landscapes for RBD monomers in the absence of disulfides. For details, see main text. (c) Ratio of intrinsic fluorescence intensity (solid red dots) at 370 nm to intensity at 335 nm as a function of time after manual-mix refolding (5 s dead time) averaged over two traces. The final protein concentration is $0.5\mu$M. The solid blue and gray lines depict the expected final ratio for RBD in the native and denatured + reduced state, respectively. The inset shows the refolding kinetics zoomed in on early times. (d) Ratio of intrinsic fluorescence intensity at 370 nm to intensity at 320 nm (black solid line) and ThT fluorescence (gray dashed line) as a function of time after reduction of RBD under native conditions. The protein and ThT concentrations are 6 and 7 $\mu M$, respectively, and the excitation and emission wavelengths used for ThT fluorescence were 450 and 485 nm, respectively.