A buried ionizable residue destabilizes the native state and the transition state in the folding of monellin.

A buried ionizable residue can have a drastic effect on the stability of a native protein, but there has been only limited investigation of how burial of an ionizable residue affects the kinetics of protein folding. In this study, the effect of burial of ionizable residues on the thermodynamics and kinetics of folding and unfolding of monellin has been investigated. The stability of wild-type (wt) monellin is known to decrease with an increase in pH from 4 to 10. The Glu24 -> Ala mutation makes the stability of the resultant E24A mutant protein independent of pH in the range from 4 to 8. An additional mutation, Cys42 -> Ala, results in the stability becoming independent of pH in the range from 4 to 10. Like the wt protein, E24A folds via very fast, fast, and slow folding pathways Compared to that of the wt protein, the rate of slow folding pathway of E24A is similar to 7-fold faster, the rate of fast folding pathway is similar to 1.5-fold faster, while the rate of very fast folding pathway is similar. E24A unfolds similar to 7-fold slower than the wt. The extent of stabilization of the transition state (TS) observed for the slow pathway of refolding and for unfolding is the same, indicating that unfolding occurs via the TS populated on the slow pathway of refolding. The stabilization of the TS of folding (1.1 kcal mol(-1)) is less than that of the native state (2.3 kcal mol(-1)) of E24A, indicating that structure has only partially formed in the vicinity of Glu24 in the TS of folding.