Figure 9. Interpretation of possible chevrons generated by the charge-burial strategy.

For an aliphatic→ charged substitution, the chevron can have a similar folding but altered unfolding arm at neutral pH. The change in slope can be used to estimate the amount of structure lost due to the substitution. If the decrease of slope is consistent with the estimate (red), it is suggestive of the creation of a folding intermediate lacking structure near the site of the substitution that otherwise is formed after the TS in the folding of the WT protein. If decrease of slope is smaller than estimation (not shown), it may be indicative of a locally frayed conformation rather than a genuine folding intermediate. Alternatively, the decrease may be consistent with the loss of an additional foldon (blue) if folding is hierarchical, being dependent on the perturbed foldon. This possibility can be tested by further characterization, e.g. with NSHX. Changes in the folding arm indicate that the TS is destabilized by the substitution. When the slope of the folding is decreased, some structure has been lost in both the TS and the ground state (cyan). More complicated scenarios also are possible.