MONITORING OXIDATIVE-DRIVEN PROTEIN FOLDING IN REAL-TIME USING SELECTIVE LABELING TOGETHER WITH FAST AND ULTRAFAST 2D NMR

Protein folding takes place on a milliseconds-to-seconds timescale. The LR5 domain from the low density lipoprotein receptor folds as controlled by the formation of disulfide bonds. LR5’s crystal structure [1] reveals three disulfide bonds and the presence of a Ca²⁺ ion, and its slow folding nature enables the use of fast NMR methods to probe this process in real-time. Indeed, although conventional 2D NMR acquisitions generally demand minutes to hours, recent developments by our and by other groups [2,3] have made it possible to acquire 2D spectra in sub-second and seconds timescales. We report here fast and ultrafast 2D NMR experiments aimed at elucidating the structural pathway of LR5’s folding; and in particular the order in which the disulfide bonds form. Intermediates along the folding pathway were observed, using selective isotope-labeling of LR5’s Cysteines and dynamic 1D ¹³C NMR. The folding intermediate formation results from closing of non-native disulfide bonds, followed by shuffling of these bonds into the native, folded conformation. However, this folding intermediate exists in several conformations, which still need to be resolved and structurally characterized. These characterizations were also observed by 2D NMR, and their full nature elucidated by higher-dimensional experiments.

1. Fass, D. et al., Nature, 1997. 388(6643): 691-693.
2. Frydman, L. et al., Proceedings of the National Academy of Sciences, 2002. 99(25): 15858-15862.
3. Schanda, P. et al., J Biomol NMR, 2005. 33(4): 199-211.